pharmaceutics-16-00947-v2

pharmaceutics
Article
Enhanced Antibacterial Activity of Clindamycin Using
Molecularly Imprinted Polymer Nanoparticles Loaded with
Polyurethane Nanofibrous Scaffolds for the Treatment of
Acne Vulgaris
Sammar Fathy Elhabal 1, *, Rehab Abdelmonem 2 , Rasha Mohamed El Nashar 3 ,
Mohamed Fathi Mohamed Elrefai 4,5 , Ahmed Mohsen Elsaid Hamdan 6, * , Nesreen A. Safwat 7 , Mai S. Shoela 8 ,
Fatma E. Hassan 9,10 , Amira Rizk 11 , Soad L. Kabil 12 , Nagla Ahmed El-Nabarawy 13 , Amal Anwar Taha 14
and Mohamed El-Nabarawi 15
1 Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Modern University for
Technology and Information (MTI), Mokattam, Cairo 11571, Egypt
2 Department of Industrial Pharmacy, College of Pharmaceutical Sciences and Drug Manufacturing,
Misr University for Science and Technology (MUST), 6th of October City 12566, Egypt;
rehab.abdelmonem@must.edu.eg
3 Chemistry Department, Faculty of Science, Cairo University, Giza 12613, Egypt; rasha.elnashar@cu.edu.eg
4 Department of Anatomy, Histology, Physiology and Biochemistry, Faculty of Medicine, The Hashemite
University, Zarqa 13133, Jordan; mohamed@hu.edu.jo
5 Department of Anatomy and Embryology, Faculty of Medicine, Ain Shams University, Cairo 11566, Egypt
6 Department of Pharmacy Practice, Faculty of Pharmacy, University of Tabuk, Tabuk 71491, Saudi Arabia
7 Department of Microbiology and Immunology, Faculty of Pharmacy, Modern University for Technology and
Information (MTI), Mokattam, Cairo 11571, Egypt; nesreen.safwat@pharm.mti.edu.eg
Citation: Elhabal, S.F.; Abdelmonem, 8 Department of Clinical Pharmacology, Faculty of Medicine, Alexandria University, Alexandria 21526, Egypt;
R.; El Nashar, R.M.; Elrefai, M.F.M.; mai.shoala@alexmed.edu.eg
9 Medical Physiology Department, Faculty of Medicine, Cairo University, Giza 11562, Egypt;
Hamdan, A.M.E.; Safwat, N.A.;
Shoela, M.S.; Hassan, F.E.; Rizk, A.; fatma.e.elsayed@kasralainy.edu.eg
10 General Medicine Practice Program, Department of Physiology, Batterjee Medical College,
Kabil, S.L.; et al. Enhanced
Jeddah 21442, Saudi Arabia
Antibacterial Activity of Clindamycin 11 Food Science and Technology Department, Faculty of Agricultural, Tanta University, Tanta City 31527, Egypt;
Using Molecularly Imprinted Polymer
amira.rizk@agr.tanta.edu.eg
Nanoparticles Loaded with 12 Department of Clinical Pharmacology, Faculty of Medicine, Zagazig University, Zagazig 44519, Egypt
Polyurethane Nanofibrous Scaffolds 13 National Egyptian Center of Environmental & Toxicological Research (NECTR), Faculty of Medicine,
for the Treatment of Acne Vulgaris. Cairo University, Cairo 11562, Egypt; n.a.nabarawy@gmail.com
14 Department of Pharmaceutics, College of Pharmaceutical Sciences and Drug Manufacturing, Misr University
Pharmaceutics 2024, 16, 947.
https://doi.org/10.3390/ for Science and Technology (MUST), 6th of October City 12566, Egypt; amal.anwar@must.edu.eg
15 Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Cairo University,
pharmaceutics16070947
Cairo 11562, Egypt; mohamed.elnabarawi@pharma.cu.edu.eg
Academic Editor: Ortensia * Correspondence: sammar_fathy2007@yahoo.com or sammar.fathy@pharm.mti.edu.eg (S.F.E.);
Ilaria Parisi a_hamdan@ut.edu.sa (A.M.E.H.)
Received: 10 May 2024

Abstract: Acne vulgaris, a prevalent skin condition, arises from an imbalance in skin flora, fos-
Revised: 9 July 2024
tering bacterial overgrowth. Addressing this issue, clindamycin molecularly imprinted polymeric
Accepted: 13 July 2024
Published: 17 July 2024
nanoparticles (Clin-MIP) loaded onto polyurethane nanofiber scaffolds were developed for acne
treatment. Clin-MIP was synthesized via precipitation polymerization using methacrylic acid (MAA),
ethylene glycol dimethacrylate (EGDMA), and azoisobutyronitrile (AIBN) as functional monomers,
crosslinkers, and free-radical initiators, respectively. MIP characterization utilized Fourier-transform
Copyright: © 2024 by the authors. infrared spectroscopy (FTIR) and transmission electron microscopy (TEM) before being incorporated
Licensee MDPI, Basel, Switzerland. into polyurethane nanofibers through electrospinning. Further analysis involved FTIR, scanning
This article is an open access article
electron microscopy (SEM), in vitro release studies, and an ex vivo study. Clin-MIP showed strong
distributed under the terms and
antibacterial activity against S. aureus, with inhibitory concentration (MIC) and minimum bactericidal
conditions of the Creative Commons
concentration (MBC) values of 0.39 and 6.25 µg/mL, respectively. It significantly dropped the bacte-
Attribution (CC BY) license (https://
rial count from 1 × 108 to 39 × 101 CFU/mL in vivo and has bactericidal activity within 180 min of
creativecommons.org/licenses/by/
4.0/).
incubation in vitro. The pharmacodynamic and histopathology studies revealed a significant decrease
Pharmaceutics 2024, 16, 947. https://doi.org/10.3390/pharmaceutics16070947 https://www.mdpi.com/journal/pharmaceutics

Pharmaceutics 2024, 16, 947 2 of 30
in infected animal skin inflammation, epidermal hypertrophy, and congestion upon treatment with
Clin-MIP polyurethane nanofiber and reduced pro-inflammatory cytokines (NLRP3, TNF-α, IL-1β,
and IL-6) conducive to acne healing. Consequently, the recently created Clin-MIP polyurethane
nanofibrous scaffold. This innovative approach offers insight into creating materials with several
uses for treating infectious wounds caused by acne.
Keywords: molecularly imprinted polymer; polyurethane nanofiber; acne; antimicrobial; clindamycin;

inflammation; Staphylococcus aureus
1. Introduction
The human body’s largest organ is the skin (1.8 m2 ); it controls body temperature,
protects important organs from harm, and more. Microbiome imbalances often cause
skin diseases such as acne vulgaris [1,2]. Acne appears as comedones, papules, pustules,
and cysts. Many factors that cause acne and other acne vulgaris bacteria have been
studied recently. The pilosebaceous unit is home to lipophilic yeasts (Pityrosporum species),
anaerobic diphtheroids (Cutibacterium granulosum and Cutibacterium acnes), and gram-
positive, coagulase-negative cocci. The main lesions of acne vulgaris, comedones, frequently
form inside sebaceous follicles. The comedone microbiome usually has a composition
similar to that of the surrounding sebaceous follicles. The etiology of acne is complex,
involving inflammation and skin microorganisms. Unusual follicular hyperkeratinization,
increased sebum production, acne colonization, and an inflammatory cascade cause acne,
which is reported to be the cause. Acne can lower self-esteem and attitudes in teens and
adults [3,4].
There are numerous approaches for treating acne vulgaris nowadays, of which topical
antibiotics such as clindamycin, erythromycin, metronidazole, retinoids, or hormones are
utilized to treat acne vulgaris. Chemical peeling agents, such as salicylic acid, glycolic
acid, and lactic acid, are also used for treatment, as they can help reduce acne lesions and
improve overall skin texture. Although acne pharmacotherapies work well, they can cause
possible side effects, such as behavioral disorders, antibiotic resistance, and dry, irritated
skin [5].
Clindamycin is a tiny antibacterial chemical agent that works well against various
pathogens, including certain protozoa, most anaerobic bacteria, staphylococci, streptococci,
and pneumococci. The bacteriostatic antimicrobial drug clindamycin inhibits ribosome
function in 50s by inhibiting bacterial protein production; clindamycin is the drug of choice
for treating infections of the skin and soft tissues [6]. Infections of the skin and soft tissues
are treated with clindamycin. It is typically applied topically for antimicrobial purposes.
Although clindamycin belongs to the Biopharmaceutics Classification System (BCS III) and
is nearly insoluble in water, its salt form, clindamycin hydrochloride (HCl), is soluble in
water and has poor permeability. Owing medications cannot naturally produce sustained
release patterns, clindamycin HCl was chosen and employed in this investigation as a
model drug of a tiny hydrophilic antibacterial compound [7].
Molecularly imprinted polymers (MIPs) represent a class of artificially designed and
synthesized polymers that use templates to recognize and upload macromolecules. The
template molecule(s) and functional monomer(s) complex are polymerized in the presence
of excess cross-linking agents that help in creating the recognition cavities to be used for
the designed purpose; MIPs can precisely rebind the template after its removal or release
based on the required application, whether it aims at selective uptake of the template as
in case of solid phase extraction, chromatographic applications, electrochemical detection,
or sustained release of such loaded templates in the case of drug delivery applications [8].
MIPs have low cost, ease of production, stability during storage, activity retention across
repeated procedures, high mechanical strength, strong thermal and chemical stability,
and applicability in aggressive media. The template and cross-linkers used in molecular
imprinting impact the polymer’s binding affinity and specificity.
Different approaches are used in molecular imprinting, the most common of which is
bulk polymerization, which is reported to be the most straightforward and most applied
technique despite its common drawbacks, including tedious washing and grinding steps
that might negatively affect the designed recognition sites. Precipitation polymerization
represents an excellent approach for nanosized molecular imprints. However, the number
of solvents used in the synthesis is almost 3–5 times that used in its bulk polymerization,
and the particles produced are of more regular size with no need for grinding or sieving [9].
Electrospinning is a simple and adaptable method for making submicron- to nanometer-
sized ultrafine fibers. This approach produces fibers with high porosities, small pore sizes,
and large surface-to-volume ratios because of their desirable properties. Electrospun
nanofibers are employed in filtration, affinity membranes, biological sciences, and optical
sensors [10].
Polymeric nanofibers have great potential in acne treatment, antimicrobial dressings,
biosensors, filtration, optoelectronics, and drug delivery. Modern techniques can be used
to fabricate nanofibers from various polymers. Examples include electrospinning, centrifu-
gal spinning, and pressured gyration. Leading nanofiber fabrication approaches include
template synthesis, phase separation, assembly, and solution blowing. Electrospinning is
the easiest and most efficient method for producing nano- to microscale fibers [11]. The
procedure uses electric fields to create nanofibers with diverse morphologies from various
polymers, composites, or mixes. Electrospun nanofibers replicate the extracellular matrix
(ECM) in structure and morphology. Additionally, its high porosity aids in nutrition trans-
port and gas penetration for developing cells. Electrospun nanofibers include a mechanical
framework that supports cell adhesion and mimics the cellular milieu. Electrospinning
produces long, continuous fibers while providing precise control over fiber dimensions,
enabling process scale [12].
Polyurethane, a thermoplastic copolymer, reacts di- or tri-isocyanate with a polyol
using a tin-based catalyst. Both long-term medical and non-medical uses use a well-
known class of elastomers. With minor alterations, medical-grade polyurethane nanofibers
have been thoroughly researched for various applications, such as tissue regeneration,
medication administration, biosensor devices, wound healing, and protective clothes.
Polyurethane nanofiber, a wound-dressing material for acne treatment, has benefits such
as water insolubility, epithelization support, non-invasiveness, and optimal pore size for
nutrition and gas mobilization. Polyurethane nanofibers’ porous nature enables gas and
nutrient exchange but prevents germs from reaching the wound surface. The hydrophobic
property of this polymer hinders skin contact, resulting in insufficient exudate adsorption
and antibacterial agent release [13].
The present study aimed to develop clindamycin antibiotics topically through molec-
ular imprinting technology; clindamycin was immobilized within the MIP via precipita-
tion polymerization, after which the resulting particles were loaded within polyurethane
nanofiber via electrospinning, followed by investigating their implementation as an effec-
tive way to treat acne bacterial infections. Nanofibers that can administer antibiotics with
prolonged release. Furthermore, hydrophilic antibiotics may have a longer release period if
the medication is encapsulated in a nanoparticle.
The morphology, physical and mechanical integrity, ex-vivo deposition and perme-
ation, thermal properties, and chemical parameters of MIP and nanofibers were investi-
gated. Furthermore, a cytotoxicity assay, inflammatory biomarker research, and Staphy-
lococcus aureus testing were performed to evaluate the anti-bacterial activity. Finally, a
histopathology investigation and an in vivo animal trial on acne were carried out, with
the assessment criterion being the reduction of inflammatory, non-inflammatory, and total
acne lesions.
2. Materials and Methods

2.1. Materials
The following products were purchased from Sigma-Aldrich (Milan, Italy): methacrylic
acid (MAA), azoisobutyronitrile (AIBN), acetone, methanol, ethylene glycol dimethacrylate
(EGDMA), and polyurethane with tetrahydrofuran (THF). All the solvents were acquired
from VWR in Milan, Italy, and were of reagent or HPLC quality. Medical International
Ltd. (London, UK) provided the dialysis membranes (molecular weight cut-off, MWCO:
3500 Da) used in the in vitro release tests.
2.2. Animals
The Cairo University Faculty of Pharmacy Ethics Committee has approved in vivo
animal research (acceptance code no. PI-3218). For in-vivo tests, eight-week-old male
Wistar albino rats weighing 150 and 200 g were split into three groups. The animals were
housed in specially designed, pathogen-free habitats with a 12-h light/dark cycle, constant
humidity, and temperatures between 20 and 24 ◦ C. They also had free reign to eat and
drink. The investigation was also carried out by ARRIVE principles, the “Guide for the
Care and Use of Laboratory Animals”, and Egyptian and local institutional legislation
about animal care, and the supervision of professional examiners.
2.3. Methods
2.3.1. Synthesis of the Clindamycin Molecularly Imprinted Polymer (Clin-MIP)
Precipitation polymerization was employed to craft a specialized MIP tailored for
clindamycin. Initially, 5 mL of methanol was utilized to dissolve the template molecule’s
424.98 mg (1 mmol) and 0.339 mL (4 mmol) of MAA in a 100 mL round-bottom flask. This
solution underwent sonication for ten minutes to foster the formation of the template-
functional monomer depolymerization complex. After adding 3.773 mL (20 mmol) of
EGDMA, 2 mL of acetone, and 150 mg of AIBN, the reaction mixture underwent an
additional ten minutes of nitrogen purging and sonication. Polymerization took place
at 60 ◦ C, with the flask rotating at 40 rpm. After 24 h, the resulting polymeric particles
were extracted via filtration, and the polymeric particles were vacuum-dried overnight at
40 ◦ C to be used for the electrospinning process. The amounts of clindamycin were tracked
using high-performance liquid chromatography (HPLC) (Agilent 1100 series, Santa Clara,
CA, USA; ACE 5 C18 HPLC column (5 µm, 150 × 4.6 mm), Scotland, with the detection
wavelength set at 210 nm. HPLC analysis was conducted using an acetonitrile phosphate
buffer solution (pH 2.5), 25:75 (v/v) in the mobile phase with a flow rate of 1.0 mL/min.
The column temperature was set at 25 ◦ C, and injections were made with a sample volume
of 20 µL. The retention time was 3.5 min. analysis to verify its purity, ensuring the absence
of other compounds, including clindamycin. Concurrently, a non-imprinted polymer (NIP)
was synthesized using the same experimental procedure as the imprinted particles, except
for omitting the template molecule [14].
2.3.2. In-Vitro Characterization of Clindamycin Molecularly Imprinted Polymer (Clin-MIP)

Particle Size and Transmission Electron Microscopy (TEM)
Using laser light scattering, the particle size, polydispersity index, and zeta potential
were measured (Malvern Zeta Sizer ZS, Malvern, Worcestershire, UK). A transmission
electron microscope (TEM) (JEM-1230, Joel, Tokyo, Japan) was employed to explore the
structure of the MIP containing clindamycin in the formulation. The samples were po-
sitioned on a carbon-coated grid, treated with a 1% phosphotungstic acid solution for
staining, and left to air dry at room temperature before observation [1].
Fourier Transformer Infrared Spectroscopy (FTIR)

A Fourier transform infrared spectrometer (Perkin-Elmer spectrum 100) was used to
investigate the interactions between clindamycin, NIP, and MIP within the formulation [15].
Discussion of Differential Scanning Calorimetry (DSC) Analysis

The thermal characteristics of clindamycin, NIP, and MIP were assessed using a
Mettler DSC 823 (Mettler Toledo, Greifensee, Switzerland) and a Julabo thermocryostate
model FT100Y (Julabo Labortechnik GmbH, Seelbach, Germany). The Mettler Star program
(version 9. x) was used for data analysis. Indium was utilized for instrument calibration.
Samples were scanned at 10 ◦ C/min in a 20–300 ◦ C temperature range [15].
Binding Studies
Static Equilibrium Adsorption Experiments
Binding studies were carried out to investigate both the recognition properties and
the selectivity of synthesized MIPs. The template was completely removed using polar
solvents such as an acetic acid–methanol mixture (1:9 v/v) for 48 h, followed by methanol
alone for another 48 h. i.e., non-covalent interactions were involved in the polymerization
process. The imprinting efficiency of the polymer was tested through incubation with
10 mg of each of the Clin-MIP and NIP in 2 mL of Clin (0.25, 0.5, 0.75, 1.0, 1.25, and 1.5 µM)
for 1 h with constant shaking. Each sample was centrifuged at 9000 rpm for 10 min, and the
Clin concentration was determined by HPLC analysis using the equation obtained from the
calibration curve of the drug. In order to investigate the selectivity of MIP, non-competitive
binding studies were also carried out in the presence of Lincomycin (Lin), which is a
structural homologue of Clin, following the same experimental procedure. The binding
experiments were repeated in triplicate.
Kinetic Adsorption Study
The adsorption kinetics of the produced particles were investigated using the following
procedure: A total of 10 mg of polymeric beads were combined with 2 mL of a standard
solution of the substance under test (Clin) with a concentration of 1 µM in a mixture of
acetic acid and methanol (1:9 v/v). The drug concentration was assessed by HPLC analysis
at various incubation durations (1–24 h), followed by centrifugation of the samples at
9000 rpm for 10 min. The concentration of Clin was measured at 210 nm using the equation
derived from the calibration curve of the medicinal drug. The experiments were conducted
three times.
2.3.3. Fabrication of Composite Polyurethane Nanofiber Dressings by Electrospinning

Unwashed Clin-MIP was encapsulated in nanofiber using an electrospinning assembly
machine (Royal HD 30, Chennai, India), and clindamycin-containing MIP powder was
co-electrospun with 14 wt.% polyurethane to create nanofibers on a molecular level. Using a
500-rpm magnetic stirrer (REMI 1MLH), the polyurethane polymer was continuously mixed
with 70 parts tetrahydrofuran (THF) during the night. First, various doses of Clin-MIP (0,
0.5, 1, 2, and 4% in DMF) were dispersed with the polyurethane polymer in THF. Three
disposable syringes (Hindustan Syringes and Medical Devices Ltd., Faridabad, Haryana,
India) with an internal needle diameter of 0.90 mm (19G) were filled with the obtained
dispersions. The made-up solutions were then poured into ten-milliliter syringes and put
within the electrospinning device holders. The fibers were pointed toward the depositing
electrode at a 115◦ angle concerning the horizontal plane. A second copper loop linked the
tips of the needles on the central syringe to a positive voltage source to guarantee an even
charge distribution. A steady voltage of 16 kV, a tip-to-collector distance of 15 cm, a flow
rate of 0.7 mL/h, a temperature of 27 ◦ C, a relative humidity of 58%, and a drum collector
speed of 140 rpm were all maintained during the electrospinning process [9]. Finally, the
nanofibers were collected on aluminum foil wrapped over the metallic drum collector with
an external diameter of 5 cm. Overall, 30 mL of the polymeric solution was deposited on
the aluminum foil, vacuum sealed, and stored for further study.
2.3.4. In-Vitro Characterization of Clin-MIP Polyurethane Nanofiber (CMPN)

Consistency in Thickness and Weight
We measured the thickness of the inserts at various points on the nanofibrous mats
using the Tork Craft Digital Micrometer 0–25 mm (ME30025). To ensure uniformity, we cut
and weighed nanofiber pieces of equal size using digital scales for each parameter. The
mean and standard deviation were then calculated and presented (mean ± SD, n = 3) [12].
Evaluation of Flexibility and Strength

To assess flexibility, the pieces were manually folded at a 180-degree angle in the
center until they tore after being cut into 2 × 2 cm2 pieces. The folding endurance was
determined by the amount of stress the fiber could withstand before rupturing. An STM-5
testing apparatus (Santam, Iran) was used to determine the skin implants’ tensile strength.
The top mobile grasp of the machine applied upward force at a rate of 1 mm/min until
the rectangular nanofiber samples (1.5–2 cm2 ) ruptured. Three duplicate measurements of
the maximum stress withstood, the percentage elongation at the breakpoint, and the break
time were made, and the results were given as means [16,17].
Fourier-Transform Infrared Spectroscopy (FTIR)

Using a Shimadzu IR PRESTIGE-21 (Japan), we examined the FTIR spectra of polyurethan
nanofiber and Clin-MIP polyurethane nanofiber samples. Samples (n = 3) were compressed
into analytical tablets using a manual compressing machine set to 9 tons of pressure after
being ground with KBr powder. The spectrometer generated FTIR spectra within the
400–4000 cm−1 wavenumber range [18].
Morphology Characterization
The shape of the nanofibrous scaffold was evaluated by SEM analysis. A Hitachi
SU3500 SEM vacuum chamber was used to examine samples coated in gold to examine the
surface characteristics of the compositions. 20 to 30 kV was the accelerating voltage [19].
Assessment of In-Vitro Drug Release

By employing the dialysis method, phosphate buffer solutions with a pH of 7.4 were
used to investigate the release pattern of the Clin, Clin-MIP, and Clin-MIP-Polyurethane
nanofibers in vitro. The nanofibers with a molecular weight cutoff of 12,000 Da were placed
into dialysis bags and submerged in 100 milliliters of the corresponding buffer solutions.
The solutions were gently stirred for 24 h at 200 rpm while kept at room temperature.
Five milliliters of the supernatant solution were removed and replaced at predetermined
intervals with an equivalent volume of brand-new buffer solution. We computed the
medication release percentage using Equation (1) [20].
Drug Release %= AR/AC × 100 (1)
In this case, AC is the total amount of clindamycin that was administered to the
nanofiber, and AR is the amount of clindamycin that was absorbed by the system, de-
termine the concentration of Clin in phosphate buffer (pH 7.4) and establish the HPLC
assay’s reliability, it was performed five times a day for five days in a row under the same
conditions, covering a concentration range of 0.05–100 µg/mL [21].
2.4. Ex Vivo Skin Permeation Study

Using Clin-MIP-Polyurethane nanofiber, we performed a skin permeation study to
examine medication penetration through the skin utilizing Franz diffusion cells. The Cairo
University Faculty of Pharmacy Ethical Committee’s (acceptance code no. PI-3218) set of
ethical principles was scrupulously followed. For the investigation, the skin of a newly
shaven rat’s abdomen was used. The stratum corneum was positioned toward the donor
compartment by attaching the skin to two vertical Franz diffusion cells, creating a 2.75 cm2
permeation region. To replicate blood pH, 100 mL of pH 7.4 phosphate buffer was added
to the receptor compartment and shaken at 100 rpm. The donor cells were supplemented
with 32 mg of Clin-MIP-Polyurethane nanofiber or 10 mg of clindamycin hydrochloride
in an exact measurement. Concurrently, the reactor compartment holding 30 milliliters
of phosphate buffered saline solution (PBS) with a pH of 7.4 was maintained at 37 ± 1 ◦ C
with magnetic stirring at 100 revolutions per minute. After six hours of removing 0.5 mL
of the permeation media, the receptor cell was filled with fresh media every hour. After
the samples were filtered through a 0.45-mm membrane, they were examined using the
previously mentioned HPLC spectroscopy procedure. The average of the three runs was
given as the result [1,22]. By charting the drug’s passage through the skin over time,
Equation (2) might be used to determine the permeability coefficient (cm/h).
Kp = Jss/CO (2)
where Jss (steady-state flux) is the slope of the linear section (mg/cm2 /h) and CO is the
initial drug concentration (mg/cm3 ). After the experiment, any remaining medication was
quickly eliminated from the skin tissues by removing them from the diffusion cell and
immersing them in pure water for ten seconds. To extract the drug that had been deposited,
the tissues were next chopped into tiny pieces and sonicated for 30 min in a bath that
contained 5 mL of methanol. Lastly, the samples were examined using the HPLC assay that
was previously explained.
2.5. In Vitro Cell Toxicity Assessment

Cell toxicity experiments conducted on human skin epithelial cells (HSE-2) assessed
the formulation’s safety. The ATCC PCS-201–012 cell line was procured from Vacsera
Egypt. Nanofiber pieces weighing 5, 2.5, and 1.25 mg were immersed in 10 milliliters
of Eagle’s modified medium with Dulbecco. Direct cell contact was allowed after 48 h
following the isolation of the supernatant. HSE-2 cells were cultured in 96-well plates
using DMEM/F-12 medium supplemented with 10% FBS, 100 units/mL penicillin, and
100 µg/mL streptomycin for 24 h at 37 ◦ C with 5% CO2 . One row served as a control with
no formulations, while each of the remaining rows received supernatants from various
formulation concentrations. DMSO was administered in a row as a control to inhibit cell
growth. After adding trypsin, the plates were washed with PBS, and the culture medium
was discarded the next day. Samples were incubated in wells with 10 µL of MTT solution
(5 mg/mL) for 4 h to assess the viability of living cells [23]. The absorbance of the samples
was measured at 570 nm, and the average of three tests was calculated. Cell viability was
determined using Equation (3).
Cell viability (%) = (sample absorbance)/(control absorbance) ×100 (3)
2.6. Determination of Antimicrobial Activity

2.6.1. Agar Well Diffusion Assay
Using the agar well diffusion assay, the antimicrobial activity of clindamycin and
clindamycin nanofiber was evaluated against standard bacteria that may be present in
the skin flora, including Streptococcus mutans ATCC 25175, Staphylococcus aureus ATCC
6538, Pseudomonas aeruginosa ATCC90274, K. pneumoniae ATCC 13883, and Proteus vulgaris
ATCC13315. The Giza, Egypt-based Egyptian Company for Biological Products and Vac-
cines (VACSERA) supplied the standard bacterial strains used in the skin profile. Using
Muller-Hinton agar (MHA) technology, 1.8 × 108 CFU/mL (0.5 OD600) of the studied
strains of bacteria were seeded onto agar plates from Oxoid, Hampshire, UK. 100 µL of a
solution containing 10 mg/mL of clindamycin and its nanofiber component dissolved in
5% DMSO was added to the 6 mm diameter wells punched in the agar plates. Additionally,
the examined plates were incubated at 37 ◦ C for 24 h, while gentamycin (Sigma, St. Louis,
MO, USA) was used as a positive control. According to CLSI guidelines, the inhibitory
zones produced after incubation were quantified in millimeters (NCCLS, 2010) [24].
2.6.2. Bacterial Proliferation Inhibition Analysis

A two-fold broth dilution method was used to determine the minimum bacterici-
dal concentration (MBC) and minimum inhibitory concentration (MIC) of Clin and Clin
nanofiber against the studied bacterial strains [4]. A 96-well microplate filled with Mullar
Hinton broth was diluted with Clin. and Clin_nanofiber, resulting in final concentrations
of 0.78, 1.56, 3.13, 6.25, 12.5, 25, and 50 µM. Each strain of bacteria under study was injected
in equal amounts (106 CFU/mL). Using a microplate reader (Infinite M1000 Pro, Tecan
Company, Männedorf, Switzerland), the absorbance of bacterial growth was measured at
600 nm following a 24-h incubation period at 37 ◦ C. The MIC values showed the lowest
concentration needed to halt growth that could be seen. The MIC test was used to calculate
the lowest bactericidal concentration (MBC).
2.6.3. Bacterial Killing Kinetic Assay

According to Wu et al., 2021, the bacterial killing kinetics of Clin_nano-formula against
acne-inducing Staphylococcus aureus ATCC 6538 were conducted [4]. Fresh Muller Hinton
broth was combined with an equal volume of bacteria and clin nanofiber at a final concen-
tration of 1× MIC. At different intervals (0, 15, 30, 45, 60, 90, 120, and 180 min), duplicate
samples containing 0.1 milliliters were taken out and spread out on agar plates. The viable
colonies were counted following a 24 h incubation period at 37 ◦ C. Additionally, a blank
containing a 1× MIC value of clindamycin was used as the positive control.
2.7. In-Vivo Study

To evaluate the anti-acne effectiveness of Clin suspension, Clin-MIP, and Clin-MIP
polyurethane nanofiber, we employed a rat model for acne in vivo. Rats demonstrate
analogous physiological processes of acne formation, making this model highly advan-
tageous for assessing the efficacy of the prepared biotherapeutic polymer scaffold on the
skin. Male Wistar albino rats weighing 150–200 g was utilized in the study. Chronic skin
inflammation was induced using Staphylococcus aureus (ATCC 6538) through intradermal
injection of bacteria into the rats’ ears. After shaving and pre-fixing with double-sided tape,
fifty microliters of bacterial cultures were intradermally injected into the backs of the rats’
left and right ears. The untreated left ear side was a control, while the right ear was treated
with formulations. Subsequently, the respective animal groups received consistent volumes
of topically applied Clin suspension, Clin-MIP, and Clin-MIP polyurethane nanofiber for
four consecutive days. The rats were divided into three groups (n = 8). All ears on the
left side acted as the control group, while the right ear side was treated as follows: Group
I received Clin suspension, Group II was treated with Clin-MIP, and Group III received
Clin-MIP polyurethane nanofiber scaffold [25].
2.8. In Vivo Evaluation of the Prepared Clin-MIP Polyurethane Nanofiber (CMPN)

2.8.1. Induction of an Inflammatory Acne Model Using the Viable Count of Infected
Rat Skin
To evaluate the in vivo anti-acne efficacy of Clin, Clin-MIP, and Clin-MIP polyurethane
nanofiber, the reduction in bacterial counts was investigated after the full treatment period
in infected rats. Inflammatory acne was induced in the rats by intradermally injecting viable
S. aureus into the right-side ears. Skin pieces from both treated and untreated (control)
ears were excised, and punch biopsies were homogenized in 20 µL of sterile saline. The
bacterial suspensions of S. aureus were then serially diluted (1:102 –1:108 ), and 0.1 mL of
each dilution was plated on a Muller-Hinton agar plate. After incubation for 24 h at 37 ◦ C,
the colony-forming units (CFUs) of S. aureus were counted. Before sacrifice, all animals
were intraperitoneally sedated with a 10% ketamine and 2% xylazine (1:1) solution at a
dosage of 0.1 mL per 100 g of body weight.
2.8.2. Assessment of Inflammatory Biomarkers

Pentobarbital sodium (200 mg/kg, IP) was used to anesthetize each rat twenty-four
hours following the prior treatment, and blood samples were taken via the retro-orbital
sinus. Then, according to the manufacturer’s recommendations, serum was tested for
tumor necrosis factor-alpha (TNF-α), leucine-rich repeat and pyrin domain-containing
protein 3 (NLRP3), nucleotide-binding oligomerization domain, IL-1β (interleukin-1 beta),
and interleukin-1 (IL-6). The enzyme-linked immunosorbent test (ELISA) kit that was used
was supplied by MyBioSource, San Diego, CA, USA.
2.9. Histopathological Examination

Skin specimens were supplied by the rat dorsum, which acted as a fixed area for all the
rats. Following a 24 h fixation in a 10% buffered formalin solution, the specimens were cut
off, washed in xylol, and dried using escalating ethanol concentrations before being embed-
ded in paraffin. Serial skin segments were sliced and layered on glass slides to a thickness
of five to seven micrometers. The tissue sections were prepared, deparaffinized with xylol,
and stained with hematoxylin and eosin (H&E) before being examined histologically under
an electric light microscope [26].
Assessment of Ear Thickness and Anti-Inflammatory Potential on the Skin’s Surface

Using the histopathological findings, scores were assessed as follows: − (no abnor-
mality), + (mild), and ++ (moderate) for alterations. Signs of inflammation were observed,
including redness, pustules, lesions, and comedones. The thickness of the degree was also
used to measure the length of the rats’ ears [27].
MAA, EGDMA, and AIBN were used as functional monomers, crosslinkers, and
radical initiators, respectively.
3. Results and Discussion

3.1. Synthesis of Clindamycin Molecularly Imprinted Polymers
Functional monomers such as MAA and EGDMA as crosslinkers and AIBN as radical
initiators play pivotal roles as crosslinkers and radical initiators in the non-covalent imprint-
ing process. During precipitation polymerization, these components generate oligomer
radicals that crosslink to form nuclei, gradually accumulating monomers and eventually
reaching a critical mass where they precipitate, forming polymeric beads. In addition to
being straightforward to prepare, compatible with high crosslinker concentrations, free
of stabilizers and additives, and able to use aprotic solvents as porogens to promote non-
covalent interactions between the template and monomer, the non-covalent imprinting
process has several benefits. The non-covalent approach is to create clindamycin-imprinted
particles because it is a commonly used technique for creating MIPs. During polymer-
ization and rebinding, this technique facilitates the creation of reversible non-covalent
contacts (ion pairs, hydrogen bonds, van der Waals forces, and dipole-dipole interactions)
between the target molecule and functional monomers. Methacrylic acid’s capacity to
create reversible hydrogen bonds with the drug molecule is why it was selected as a func-
tional monomer. Meanwhile, EGDMA enhanced the polymer’s mechanical strength and
preserved the monomers’ functions surrounding the template molecule and recognition
cavity architecture, which helps in the release of clindamycin from the polymeric matrix.
MAA and EGDMA, being biocompatible polymers, have a well-established track record
in pharmaceutical dosage form, drug delivery, nanotechnology, and biomedical polymer
synthesis [28,29].
3.2. In-Vitro Characterization of Clindamycin Molecularly Imprinted Polymer (Clin-MIP)

3.2.1. Particle Size and Transmission Electron Microscopy (TEM)
DLS was used to analyze particle size and polydispersity index, as shown in Table 1.
The decreased size of NIP nanoparticles compared to MIP nanoparticles may be due to
clindamycin and hydrogen interaction in the reaction medium, MAA functional monomer.
The hydrogen interaction affects particle nucleation and growth, making MIP nanoparti-
cles larger than NIP ones. Additionally, decreasing cross-linker or increasing functional
monomer concentration results in smaller particles.
Table 1. (a). Formulations, particle size, and polydispersity index Yield of Clin-Imprinted and
Non-Clin Imprinted Polymers. (b). The percent of Clindamycin (Clin) and Lincomycin (Lin) that
are bound by imprinted (MIP) and non-imprinted (NIP) particles, as well as the α and ε values for
various Ci concentrations. The data are presented as the mean ± S.D. (c). Values of Ka , Bmax , and R2
by Scatchard analysis. (d). Kinetics data for Clin-MIP and Clin-NIP.
a
Clindamycin MAA AIBN EGDMA Particle Size Polydispersity Index
Sample
Template (mg) (mmol) (mg) (mmol) (nm) (PdI)
NIP 1 0 2 150 10 84 0.56
NIP 2 0 4 150 20 45 0.51
MIP 1 424.98 2 150 10 150 0.44
MIP2 424.98 4 150 20 110 0.49
b
Bound Clin (%) Bound Lin (%)
Ci (mol/L) α Clin α Lin ε
MIP NIP MIP NIP
0.25 48 ± 0.31 39 ± 0.61 18 ± 0.30 17 ± 0.60 1.23 1.06 2.67
0.5 59 ± 0.61 43 ± 0.31 20 ± 0.60 18 ± 0.30 1.37 1.11 2.95
0.75 67 ± 0.21 49 ± 0.12 24 ± 0.20 21 ± 0.10 1.37 1.14 2.79
1 79 ± 0.21 51 ± 0.01 26 ± 0.20 22 ± 0.01 1.55 1.18 3.04
1.25 78 ± 0.21 57 ± 0.01 31 ± 0.20 29 ± 0.01 1.37 1.07 2.52
c
High-Affinity Sites Low-Affinity Sites
Polymer
Ka (M−1 ) Bmax (mM/g) R2 Ka (M−1 ) Bmax (mM/g) R2
MIP 0.7347 0.1123 0.933 0.0891 0.1642 0.8845
NIP 0.1469 0.0197 0.9495
d
Pseudo-First Order Pseudo-Second Order
Polymer Qe K1 Qe K2
R2 R2
(mol/g) (h−1 ) (g/mol/h) (h−1 )
MIP 0.0003286 0.1309 0.9994 −3.718 × 10−6 6.42 × 1011 0.9842
NIP 0.0007199 0.0181 0.9995 4.06 × 109 −0.000806 0.9885
TEM imaging was used to characterize the enhanced clin-MIP formulae and MIP
without clindamycin, as shown in Figure 1a. Photos showed A non-uniform distribution
of monomers and crosslinkers polymerizes forming flower-shaped nanoparticles with a
crystallite size of around 25–45 nm. Polymeric flower beads form when nuclei expand
and grab monomers and oligomers until they reach critical nanoparticles. The in vitro
release profile showed that irregular, flower-shaped nanoparticles released clindamycin
well. The particle shape affects particle surface-medium interactions, which is critical for
the regulated release of encapsulated contents, according to the literature [30,31].
Pharmaceutics 2024, 16, x FOR PEER REVIEW 11 of 32
MIP 0.0003286 0.1309 0.9994 −3.718 × 10−6 6.42 × 1011 0.9842

NIP 2024, 16,
Pharmaceutics 0.0007199
947 0.0181 0.9995 4.06 × 109 −0.000806 0.9885 11 of 30
(a)
(b)
Figure 1. Cont.
Pharmaceutics 2024,16,
2024,
Pharmaceutics 16,947
x FOR PEER REVIEW 13 of 32 12 of 30
(c)
(d)
Figure 1. Cont.
Pharmaceutics 2024,16,16,
Pharmaceutics 2024, 947 PEER REVIEW
x FOR 14 of 32 13 of 30
(e)
Figure 1. (a):
Figure (a): Transmission
1. Transmission Electron
Electron Microscopy
Microscopy (TEM) imaging
(TEM) imaging of (I) molecularly
of (I) molecularly imprintedimprinted
poly- polymer
mer(Clin-MIP)
(Clin̶MIP) and
and(II) Non-molecularly
(II) Non-molecularly imprinted polymer
imprinted without
polymer clindamycin
without (NIP); (b):(NIP);
clindamycin (I) Fou-
(b): (I) Fourier
rier Transformer Infrared Spectroscopy (FTIR) (II) Discussion of Differential Scanning Calorimetry
Transformer Infrared Spectroscopy (FTIR) (II) Discussion of Differential Scanning Calorimetry for
for Non-imprinting polymer (NIP), Clindamycin (Clin), and Clindamycin molecularly imprinted
Non-imprinting
polymer polymer (NIP),
(Clin̶MIP), respectively. Clindamycin
(c): Adsorption (Clin),
isotherms and
of (I) Clindamycin
Clindamycin molecularly
(Clin) imprinted poly-
and (II) Linco-
mer(Lin)
mycin (Clin-MIP), respectively.
on imprinted (c): Adsorption
and non-imprinted isotherms
particles and theirof (I) Clindamycin
chemical structures.(Clin) and (II) Lincomycin
(d) Scatchard
analysis.
(Lin) (e)
on Adsorption
imprinted andkinetic curves for Clin̶particles
non-imprinted MIP and Non-Imprinted
and their chemicalPolymer (Clin̶NIP).
structures. (d) Scatchard analysis.
(e) Adsorption kinetic curves for Clin-MIP and Non-Imprinted Polymer (Clin-NIP).
3.2.2. Fourier Transformer Infrared Spectroscopy (FTIR)
3.2.2.
FigureFourier Transformer
1bI shows the FTIR Infrared
data that Spectroscopy
provided evidence (FTIR)for the production of Clin
and Clin-MIP. At frequencies of 3306 cm −1 (O-H stretching vibrations), 2891 cm−1 (C-H
Figure 1bI shows the FTIR data that provided evidence for the production of Clin
stretching vibrations),
and Clin-MIP. At 990 cm−1, and of
frequencies 1900 cmcm
3306
−1 (C-O stretch),
−1 (O-H a substantial
stretching absorption
vibrations), 2891of cm−1 (C-H
Clin or Clin-MIP was observed in the − FTIR spectra. MAA,
1 − 1 EGDMA, and AIBN were used
stretching vibrations), 990 cm , and 1900 cm (C-O stretch), a substantial absorption of
as functional monomers, crosslinkers, and radical initiators, respectively, as revealed by a
Clin or Clin-MIP was observed in the FTIR spectra. MAA, EGDMA, and AIBN were used
characteristic band at 1190 cm−1. The cross-linking group’s C=O stretching vibrations were
as functional
identified monomers,
as the source of this crosslinkers,
band. andacid
Carboxylic radical initiators,
moieties causedrespectively, as at
the absorption revealed by a
characteristic band at 1190 − 1
cminteract
. Thewith
cross-linking group’s C=O
1724 cm−1 [6,28]. Melatonin did not Clin and Clin-MIP in anystretching vibrations were
of the synthe-
identified
sized samples, as
butthe source
all had of this
similar band.
spectra withCarboxylic
similar bands acid moieties caused
at distinctive the absorption at
wave numbers
and1724 cm−intensities.
relative
1 [6,28]. Melatonin did not interact with Clin and Clin-MIP in any of the synthe-
The presence of carboxylic groups’ OH groups in the Clin and
sized samples,
Clin-MIP was ascribedbuttoallthe
had similar of
expansion spectra with similar
the absorption bandbands at distinctive
in the 3300–2810 cm−1wave
re- numbers
gion.
andThe shape and
relative position ofThe
intensities. the presence
peaks of NIPs are very similar
of carboxylic groups’to MIP
OHwith Clin. in
groups The the Clin and
creation of thewas
Clin-MIP delivery systems
ascribed required
to the an understanding
expansion of the interactions
of the absorption band in the 3300–2810 cm−1
between
polymers
region.andThedrugs.
shapeThus,
and it was imperative
position to employ
of the peaks of NIPscomputational tools to
are very similar to examine
MIP with Clin. The
these connections
creation of thesimultaneously
delivery systems [32]. required an understanding of the interactions between
polymers and drugs. Thus, it was imperative to employ computational tools to examine
3.2.3. Discussion of Differential Scanning Calorimetry (DSC) Analysis
these connections simultaneously [32].
The DSC analysis provides valuable insights into the thermal properties and physical
state. Amorphous
3.2.3. polymers
Discussion have a glass
of Differential transition
Scanning temperature(DSC)
Calorimetry of 60 °C, as shown by the
Analysis
DSC analysis of the NIP as shown in Figure 1bII. The absence of a melting peak shows the
NIP has The DSC analysis
few crystalline provides
areas. valuable
NIP thermal insights
stability is shownintobythe thermal
thermal propertiesatand physical
deterioration
◦ C, as shown by the
240 °C. This shows that the NIP is mostly amorphous and stable under test60
state. Amorphous polymers have a glass transition temperature of conditions.
DSC
Pure analysis of
Clindamycin hasthe NIP asmelting
a strong shownpeakin Figure
at 150 1bII.
°C in The absence
the DSC of a melting
thermogram, peak shows the
showing
NIP has few
crystallinity. Pure,crystalline areas. NIP thermal
crystalline compounds stability melting
have well-defined is shown by thermal
points. deterioration at
Understand-
◦ C. This shows that the NIP is mostly amorphous and stable under test conditions. Pure
ing240
Clindamycin’s physical condition before and after polymer matrix incorporation re-
quires this information.
Clindamycin has a Clin-MIP has 150peak
strong melting °C onatits150
DSC ◦ Cthermogram, somewhat higher
in the DSC thermogram, showing crys-
than NIP (50 °C).
tallinity. Pure,Clindamycin
crystallinemay interact with
compounds the well-defined
have polymer matrixmelting
due to the successful
points. Understanding
Clindamycin’s physical condition before and after polymer matrix incorporation requires
this information. Clin-MIP has 150 ◦ C on its DSC thermogram, somewhat higher than NIP
(50 ◦ C). Clindamycin may interact with the polymer matrix due to the successful creation
of molecularly imprinted sites as the transition temperature increases. The Clin-MIP’s Clin-
damycin melting peak is absent or reduced, indicating that the molecules are thoroughly
integrated into the polymer matrix, disturbing their crystalline form. This disruption
indicates successful molecular imprinting, where the template molecule (Clindamycin)
alters the polymer’s thermal characteristics [33]. Clin-MIP degrades at 300 ◦ C, somewhat
higher than NIP (270 ◦ C), suggesting that Clindamycin slightly improves polymer thermal
stability. Clindamycin and the polymer matrix may interact to improve heat stability and
structural stability.
3.2.4. Binding Studies

Imprinting Efficacy and Selectivity
Understanding the adsorption isotherms enables one to characterize the nonlinear
and dynamic equilibrium that exists between the quantity of template in the solution
and the quantity of template adsorbed on the polymeric matrix. To further optimize the
utilization of molecularly imprinted polymers, isotherm data analysis is a critical step. A
curve that connects the equilibrium concentration of the same analyte in solution (Ce) to
the amount of an analyte adsorbed onto the polymer at equilibrium (Qe), represented in
this case by the template molecule, is known as an adsorption isotherm. Based on the kind
of adsorption process that takes place, the Qe/Ce relationship can change. As a result, one
can obtain important information on the template polymer interaction through adsorption
isotherms, which can look different depending on the model used. The amount of Clin
bound to the polymers at equilibrium (Qe, mol/g) may be obtained from the binding
experiments carried out, allowing us to calculate the binding capacity of MIP and NIP
particles Equation (4) [34].
(Ci − Ce) × V
Qe = (4)
m
The solution’s volume is denoted by V (L), the weight of the polymers is indicated
by m (g), and the initial and equilibrium Clin concentrations are indicated by Ci and Ce
(mol/L), respectively.
By plotting Qe versus Ci, the adsorption isotherms of Clin on MIP and NIP particles
were obtained (Figure 1c). The outcomes of the current research investigation have verified
that the imprinted particles possess the capability to bind a greater quantity of medication
in comparison to the matching non-imprinted particles. In addition, as the initial con-
centration of Ci in the Clin grew, the adsorption capabilities of both polymeric materials
likewise increased until reaching a saturation point. Lincomycin (Lin) standard solutions at
different concentrations were used to conduct non-competitive binding tests. The purpose
of these tests was to investigate the cross-reactivity of MIP in the presence of a structural
equivalent of Clin (Figure 1c). The data in Table 1b indicates the efficacy of the imprinting
procedure and the selective binding ability of the produced imprinted particles to their
target molecules, as demonstrated by the percentages of bound Clin and Lin. The binding
sites present in the imprinted polymers were created during the polymerization process,
which took place in the presence of the specific medicinal drug. Conversely, non-specific
interactions account for the quantity of Lin that is bound to both polymeric matrices. The
imprinting factor (α) and the selectivity coefficient (ε) were calculated for the synthesized
polymeric materials, and the findings are presented in Table 1b. The initial parameter
signifies the ratio of the analyte (either the template or its counterpart) absorbed by MIP
to the analyte absorbed by NIP. The second parameter is determined by calculating the
ratio of Clin to Lin adsorbed by MIP. The α and ε values provided evidence of the selective
recognition capabilities of the imprinted material for the therapeutic drug, as opposed
to the similar non-imprinted material. The α Clin values found for each adopted Ci are
greater than 1.23, indicating that the MIP has a superior ability to bind the therapeutic drug
compared to a similar non-imprinted polymer. Furthermore, the imprinted particles exhibit
selectivity for Clin that is 2.52 to 3.04 times higher, as indicated by the measured ε values.
To better understand the adsorption properties of the synthesized MIP and the distribution
of affinity sites, the binding data was analyzed using the Scatchard model equation (5) [35].
Qe
= (Bmax − Qe)Ka (5)
Ce
This model helps distinguish between homogeneous and heterogeneous sites. In
the equation, Qe represents the amount of Clin bound per gram of polymeric material at
equilibrium (mol/g), Ce is the drug concentration at equilibrium (mol/L), Bmax (mM/g) is
the maximum binding capacity, and Ka (M−1 ) is the association constant. Thus, the binding
parameters Ka and Bmax were determined by graphing Qe/Ce against Qe, with the slope
representing Ka and the intercept representing Bmax . These estimates were obtained from
Figure 1d and Table 1c. Figure 1d demonstrates that the relationship between Qe/Ce and
Qe in MIP is not linear. Instead, it consists of two distinct straight lines with varying slopes.
This suggests that the imprinted particles have heterogeneous binding cavities, which can
be categorized into high-affinity and low-affinity sites. In contrast, the NIP Scatchard plot
consists of a solitary line, showing the existence of only one type of binding site.
Adsorption Kinetics
Adsorption kinetics refers to the measurement of the rate at which adsorption occurs
over time while maintaining a constant concentration of the medication. The current study
investigated the kinetics of adsorption by submerging 10 mg of the polymeric particles
in 2 mL of a Clin standard solution (1 µM) and measuring the drug concentration at
various time intervals. The quantity of a therapeutic agent that was attached at time t
(Qt, mol/g) was calculated by subtracting the initial concentration of Clin at t = 0 (Ci,
mol/L) from the remaining concentration at the adsorption time t (Ct, mol/L), as described
in Equation (6) [35].
V
Qt = (Ci − Ct) (6)
M
The variable V (L) represents the volume of the incubation solution, while m (g)
represents the quantity of polymeric material. Figure 1e displays the adsorption kinetic
curves of the synthesized imprinted and non-imprinted particles, represented by the plot
of Qt against t. The process of adsorption achieved a state of fast equilibrium within the
initial 10 h, followed by a slower phase until it eventually reached a state of equilibrium.
At this point, a plateau phase was seen. Moreover, the adsorption capacities of non-
imprinted particles were consistently lower than those of comparable imprinted particles at
all time points. Following the initial hour, approximately half (50%) of the clindamycin was
absorbed by MIP. The absorption levels increased gradually over time, reaching 66.69%,
78.63%, 83.73%, 90.55%, 90.80%, 90.89%, and 91.06% at the time intervals of 1, 4, 6, 8, 12,
24, 28, and 30 h, respectively. However, the quantity of clindamycin that was attached to
the particles without imprinting was 30% within the first hour. This amount increased
to 49.63%, 49.63%, 50.51%, 50.55%, 50.51%, 50.85%, and 50.85% at the time intervals of
10, 12, 16, 20, 24, 28, and 30 h, respectively. The collected experimental data was used to
fit kinetic models of pseudo-first order and pseudo-second order to examine the kinetics
of Clin adsorption on the produced MIP. The pseudo-first-order or Lagergren model is
expressed by the following Equation (7) [34]:
K1
log(Qe − Qt) = log(Qe) − t (7)
2.303
The variable Qe indicates the quantity of adsorbed Clin at the point of equilibrium,
while K1 denotes the rate constant for first-order adsorption and t represents the duration
of adsorption. This model operates on the assumption that a single molecule is adsorbed
within the active site of the polymer. Furthermore, it assumes that the process of drug
adsorption, which is regulated by the diffusion step, falls under the category of physical
adsorption. Equation (8) describes the pseudo-second-order model.
t 1
= (8)
Qt K2 Qe2
K2 represents the rate constant for adsorption in a pseudo-second-order reaction. This

kinetic model postulates that a single adsorbate molecule interacts with two active sites
and that the adsorption process is a chemical process, which may be the step that limits the
rate. The kinetic fitting data for the imprinted and non-imprinted particles are documented
in Table 1d. An analysis was conducted to determine the suitability of the two kinetic
models for describing the adsorption behavior of MIP and NIP. This analysis examined
the correlation coefficients (R2 ) as well as the experimental and estimated binding capacity
(Qe). The adsorption process exhibited pseudo-first-order kinetics for both the imprinted
and non-imprinted materials, as indicated by the higher R2 values. Furthermore, the Qe
values calculated using the pseudo-first-order kinetic model were more accurate compared
to the Qe values obtained from the pseudo-second-order equation, indicating that the latter
was not appropriate for the experimental conditions used. In addition, the larger MIP
K1 value indicates that the imprinted particles have a noticeably faster adsorption rate
compared to the non-imprinted particles. As observed from the results for MIP and NIP,
both models fit the data well, but MIP exhibits a higher adsorption rate than NIP based on
the pseudo-first-order kinetic model rate constants (K1 ).
3.3. Fabrication of Composite Polyurethane Nanofiber Dressings by Electrospinning

This study investigated polyurethane nanofibers loaded with Clin-MIP and compos-
ites as antibacterial dressings for treating acne. Several trial tests were conducted with
different voltages, flow rates, needle-to-collector distances, polymer weights, and Clin-MIP
concentrations to determine the ideal polyurethane concentration for bead-free fibers. These
screening studies discovered fibers with unusual aggregation patterns, such as beaded,
distorted, or both. As mentioned in the technique section, bead-free fibers were produced
with a polyurethane content of 14% (w/w) and a solvent ratio of 70:30 (THF: DMF). For elec-
trospinning, Clin-MIP in DMF at a 4% polyurethane concentration was selected. Beyond
this range, the viscosity of the polyurethane solution decreased, and the rate of dripping
increased with the clindamycin ions in the clindamycin salt [36].
3.4. In-Vitro Characterization of Clin-MIP Polyurethane Nanofiber (CMPN)

3.4.1. Thickness and Weight Uniformity
Test results for thickness and weight homogeneity are in Table 2. Nanofiber pieces
with identical sizes had a mean weight of 31.54 ± 0.1 to 22.65 ± 0.2 mg. The SD was less
than 0.7% of the mean weight values. Indicated formulation uniformity. A higher thickness
was observed in Clin-MIP polyurethane nanofiber (0.28 ± 0.01) compared to polyurethane
nanofiber (0.18 ± 0.01). The nanofibrous mat thickness was uniform throughout both
formulations. The right thickness and weight of the prepared dressing make it easy to
use [13,28].
Table 2. Physicochemical characteristics of nanofibers (mean ± SD).
Weight Uniformity Thickness Folding Endurance Tensile Strength

Formulation
(mg) (mm) (Times) (MPa)
Clin-MIP-polyurethane nanofiber 31.54 ± 0.1 0.28 ± 0.01 174 ± 3 1.82 ± 0.03
polyurethane nanofiber 22.65 ± 0.2 0.18 ± 0.01 154 ± 5 1.99 ± 0.02
3.4.2. Flexibility and Strength Testing Pieces of Nanofiber

One popular test for assessing dressing implant flexibility is folding endurance, as
shown in Table 2. Nanofibers showed a folding endurance ranging from 154 ± 5 to
174 ± 3 times. Both inserts appear strong enough to resist ripping after being placed in the
skin. For optimal folding endurance, fibers should not irritate the skin or dissolve quickly
in the skin. Both implants showed strong tensile strength for skin use. Table 2 data indicates
that Clin-MIP polyurethane nanofiber (CMPN) has lower strength, elongation, and break
time than Clin-MIP polyurethane nanofiber (CMPN). The higher inherent flexibility of
Clin-MIP polyurethane nanofiber compared to polyurethane fiber may explain the higher
strength of Clin-MIP polyurethane nanofiber compositions [17].
3.4.3. Morphology Characterization of Clin-MIP Polyurethane Nanofiber

A scanning electron microscope (SEM) measures the morphology and diameter of
silk fibers. On the other hand, the SEM pictures of the Clindamycin-MIP-polyurethane
nanofibers show a fibrous morphology that is comparable to that of the pristine polyurethane
nanofibers. This suggests that adding molecularly imprinted polymer (MIP) did not
substantially change the nanofibers’ overall structure. Nevertheless, the performance
and functionality of the nanofibers may be impacted by minute alterations in the sur-
face morphology and porosity brought about by the presence of clindamycin-MIP in the
polyurethane matrix. The produced fibers had continuous and homogeneous structures.
Clin-MIP polyurethane nanofiber (CMPN) had random nanofiber alignment, with a mean
diameter of 411 ± 3.32 nm, as shown in Figure 2a,b. When fiber diameter
Pharmaceutics 2024, 16, x FOR PEER REVIEW 19 ofincreases,
32 tensile
strength and modulus improve.
SEMSEM
Figure2. 2.
Figure images
images of Clin̶
of silk (a) silkM(a) Clin-MIP polyurethane
IP polyurethane nanofiber (CMPN),nanofiber (CMPN),
(b) Polyurethane (b) Polyurethane
nano-
fiber (PN), and FTIR of PN and CMPN, (c) Polyurethane nanofibers’ FTIR spectra.
nanofiber (PN), and FTIR of PN and CMPN, (c) Polyurethane nanofibers’ FTIR spectra.
3.5. In-Vitro Drug Release

Clindamycin release patterns from MIP and MIP-polyurethane nanofibers were
measured and contrasted with those from the clindamycin solution shown in Figure 3.
All formulations in this study contain 424.98 mg clindamycin, 4 mmol MMA, 150 mg
AIBN, and 20 mmol EGDMA. Anticipatedly, during the first hour of the trial, 40 ± 0.87%
Figure 2c shows Polyurethane nanofibers’ FTIR spectra show distinctive absorption

bands indicative of the functional groups found in the polymer chain. The stretching
vibrations of N-H and O-H groups correlate to the peaks at around 3300–3500 cm−1 ,
suggesting the presence of urethane and hydroxyl functionalities. The absorption bands
located about 1700–1750 cm−1 are ascribed to the carbonyl groups in the urethane linkage’s
C=O stretching vibrations. Furthermore, the C-O stretching vibrations of the ether links in
the polymer backbone correspond to maxima at about 1200–1250 cm−1 .
Similar absorption bands corresponding to polyurethane functional groups are seen
in the FTIR spectra of the clindamycin-MIP-polyurethane nanofibers, suggesting that the
polymer structure is retained following the inclusion of molecularly imprinted polymer
(MIP). Comparing the FTIR spectra of polyurethane nanofibers with Clindamycin-MIP-
polyurethane nanofibers sheds light on the materials’ structural and chemical makeup. The
presence of clindamycin-MIP may cause changes to certain functional groups or bonding
interactions within the polymer matrix, even when the overall spectral characteristics stay
the same. These modifications can signify the integration of clindamycin-MIP and its 20 of 32
Pharmaceutics 2024, 16, x FOR PEER REVIEW
possible impact on the physicochemical characteristics of the nanofibers.
3.5. In-Vitro Drug Release

advantageous
Clindamycin forrelease
the dermatological treatment
patterns from MIP of acne should nanofibers
and MIP-polyurethane they concentrate within the
were mea-
skin’s follicular
sured and openings
contrasted following
with those topical
from the application
clindamycin [20,37].
solution shown in Figure 3.
Figure 3.3. Release

Figure Releaseprofiles of of
profiles clindamycin fromfrom
clindamycin Clin-MIP and Clin-MIP
Clin-MIP polyurethane
and Clin-MIP nanofibers
polyurethane nanofibers
compared to a clindamycin solution.
compared to a clindamycin solution.
All formulations in this study contain 424.98 mg clindamycin, 4 mmol MMA, 150 mg
3.6. Ex-Vivo Skin Permeation Study
AIBN, and 20 mmol EGDMA. Anticipatedly, during the first hour of the trial, 40 ± 0.87%
of theFranz diffusion
medication fromcells
the combined
drug solution with
wasratreleased,
skin as and
a permeation
by the fourthmembrane were used
hour of the
in
experiment, 98 ± 2% had been
ex-vivo permeation investigations.
released. OnAlltheformulations
other hand, thein this study
release contain could
of clindamycin 424.98 mg of
be considerably4 controlled
clindamycin, mmol of MMA, (p < 0.05) bymg
150 the of
nanoparticles
AIBN, andat20allmmol.
time points, irrespective of
As a permeation medium
phosphate buffer (pH 7.4) was employed. At 24 h, clindamycin from formulations
their MIP—polyurethane nanofiber. The drug release characteristics of the two Clin-MIP exhibited
were rather close to one another, releasing only roughly 77% and 50% of the clindamycin
less skin penetration than Clin-MIP-Polyurethane nanofiber (Figure 4; Table 2); the steady
from Clin-MIP and Clin-MIP-Polyurethane nanofiber, respectively, after 30 h. NIP exhibits
state flux and permeability coefficient are shown in Table 3. Polyurethane has been shown
the least amount of drug release, suggesting a reduced affinity for the medication and
to reduce
a lack the drug’s
of binding sites.crystallinity,
30 h of releasewhich increases
of 38 the amount
µg is significantly ofthan
less drugthat
released
of MIPfrom the
nanofiber as concentration rises [1,21]. MAA was utilized as a monomer
and MIP-nanofiber. This property will enable MIP to have a rapid onset of action; this since it diffuses
and softens
variation the polymer
releases matrix.
the drug more In addition
quickly than theto creatingtype
nanofiber a link
andwith othersystems,
nanofiber polymer mole-
cules that extending
potentially aid in creating MIP-polyurethan
the duration nanofiber,
of the therapeutic it lessens
impact, which would the
beinteractions
advantageousbetween
polymer molecules, such as hydrogen bonding. An analysis was also completed on the
impact of particle size on permeation. The findings demonstrated that the degree of drug
penetration through the stratum corneum increased with decreasing particle size; the
smaller the particle size, the higher the surface of the particles and the more intimate their
contact with corneocytes. Analysis of the impact of surfactant on permeation revealed that
a rise in the ratio of surfactant to lipid corresponded to an increase in medication penetra-
for the dermatological treatment of acne should they concentrate within the skin’s follicular
openings following topical application [20,37].
3.6. Ex-Vivo Skin Permeation Study

Franz diffusion cells combined with rat skin as a permeation membrane were used
in ex-vivo permeation investigations. All formulations in this study contain 424.98 mg of
clindamycin, 4 mmol of MMA, 150 mg of AIBN, and 20 mmol. As a permeation medium,
phosphate buffer (pH 7.4) was employed. At 24 h, clindamycin from Clin-MIP exhibited
less skin penetration than Clin-MIP-Polyurethane nanofiber (Figure 4; Table 2); the steady
state flux and permeability coefficient are shown in Table 3. Polyurethane has been shown
to reduce the drug’s crystallinity, which increases the amount of drug released from the
nanofiber as concentration rises [1,21]. MAA was utilized as a monomer since it diffuses
and softens the polymer matrix. In addition to creating a link with other polymer molecules
that aid in creating MIP-polyurethan nanofiber, it lessens the interactions between polymer
molecules, such as hydrogen bonding. An analysis was also completed on the impact of
particle size on permeation. The findings demonstrated that the degree of drug penetration
through the stratum corneum increased with decreasing particle size; the smaller the
particle size, the higher the surface of the particles and the more intimate their contact with
corneocytes. Analysis of the impact of surfactant on permeation revealed that a rise in the
ratio of surfactant to lipid corresponded to an increase in medication penetration through 21 of 3
Pharmaceutics 2024, 16, x FOR PEER REVIEW
the skin. The surfactant may facilitate medication penetration by loosening the stratum
corneum’s lipid bilayers [38,39].
Figure4.4. Ex-vivo
Figure Ex-vivo skin
skinpenetration
penetrationofofclindamycin
clindamycin from
from Clin-MIP
Clin-MIP andand Clin-MIP
Clin-MIP polyurethane nan
polyurethane
ofiber compared
nanofiber comparedtotoaaclindamycin solution.
clindamycin solution.
Table 3. Ex-vivo pharmacokinetic parameters of clindamycin from Clin-MIP and Clin-MIP

3.7. In Vitro Cell Toxicity
polyurethane nanofibers compared to a clindamycin solution estimated by linear regression.
Using HSE-2 cells, the safety of clindamycin loaded with MIP polyurethane nano
Formula
fiber was investigated.
Jss The results
Kp are shown in Figure
Q24 5 for a full day
Skin of incubation; con
Deposition
(µg/cm 2 /h) (cm/h) 2) 2)
centrations of clin-MIP polyurethane nanofiber (µg/cm
up to 450 μg/mL were(µg/cm
incubated withou
Clin suspension 15 ± 0.21 1.5 ± 0.11 450 ± 0.43 123.75 ± 0.64
producing any unfavorable side effects. More than 90% of the cells were living cells. Thus
Clin-MIP it can be12.78
concluded
± 0.32 that the1.28 Clin-MIP
± 0.42 polyurethane nanofiber solution
620 ± 0.45 169.75 ±showed
0.55 no nega
tive effects
Clin-MIP polyurethane nanofiber 19.44at±any
0.33 of the evaluated concentrations.
0.6075 ± 0.41 830 ±Clindamycin
0.13 MIP is ±
171.015 included
0.23 in poly
urethane nanofibers to provide controlled release of the medication over a longer dura
tion. Clindamycin’s cytotoxic effects on cells are reduced by this controlled release profile
which aids in maintaining therapeutic concentrations of the drug. The remarkable cel
survivability may have been further attributed to optimizing the nanofiber construction
technique, which guaranteed consistent medication distribution and minimum damag
to encapsulated cells during manufacturing. The high cell viability (>85%) seen in the cy
totoxicity assessment of the clindamycin MIP-loaded polyurethane nanofibers is a resul
of a combination of biocompatible materials, molecular imprinting technology, regulated
drug release, and an optimized production process [40]. This indicates the formulation’
Figure 4. Ex-vivo skin penetration of clindamycin from Clin-MIP and Clin-MIP polyurethane nan
ofiber compared to a clindamycin solution.
Pharmaceutics 2024, 16, 947 3.7. In Vitro Cell Toxicity 20 of 30
Using HSE-2 cells, the safety of clindamycin loaded with MIP polyurethane nano
fiber was investigated. The results are shown in Figure 5 for a full day of incubation; con
3.7. In Vitro Cell Toxicity
centrations of clin-MIP polyurethane nanofiber up to 450 μg/mL were incubated withou
Using HSE-2 cells, the safety of clindamycin loaded with MIP polyurethane nanofiber
producing any unfavorable side effects. More than 90% of the cells were living cells. Thus
was investigated. The results are shown in Figure 5 for a full day of incubation; concen-
it can beofconcluded
trations that the Clin-MIP
clin-MIP polyurethane nanofiber polyurethane nanofiber
up to 450 µg/mL weresolution
incubatedshowed
withoutno nega
producing any unfavorable side effects. More than 90% of the cells were living cells. Thus, in poly
tive effects at any of the evaluated concentrations. Clindamycin MIP is included
urethane
it nanofibers
can be concluded thattotheprovide
Clin-MIPcontrolled
polyurethane release of the
nanofiber medication
solution showedover a longer dura
no negative
effects at any of the evaluated concentrations. Clindamycin MIP is included in polyurethane
tion. Clindamycin’s cytotoxic effects on cells are reduced by this controlled release profile
nanofibers
which aidstoin provide controlled
maintaining release of the
therapeutic medication over
concentrations a longer
of the drug.duration. Clin-
The remarkable cel
damycin’s cytotoxic effects on cells are reduced by this controlled release profile,
survivability may have been further attributed to optimizing the nanofiber construction which aids
in maintaining therapeutic concentrations of the drug. The remarkable cell survivability
technique, which guaranteed consistent medication distribution and minimum damage
may have been further attributed to optimizing the nanofiber construction technique, which
to encapsulated cells during manufacturing. The high cell viability (>85%) seen in the cy
guaranteed consistent medication distribution and minimum damage to encapsulated cells
totoxicity
during assessment The
manufacturing. of the
highclindamycin
cell viabilityMIP-loaded
(>85%) seen in polyurethane
the cytotoxicity nanofibers
assessment is a resul
of the
of a combination
clindamycin of biocompatible
MIP-loaded materials,
polyurethane molecular
nanofibers is aimprinting technology,of
result of a combination regulated
drug release, materials,
biocompatible and an optimized
molecular production process [40].
imprinting technology, This indicates
regulated drug release,theand
formulation’s
an
potential production
optimized in biomedical applications
process requiring
[40]. This indicates theminimum cytotoxicity
formulation’s potential inand sustained drug
biomedical
applications
delivery, suchrequiring minimum
as in tissue cytotoxicity
engineering and sustained
clindamycin drug delivery,
delivery systems such as inlocalized
or the tissue treat
engineering clindamycin delivery systems or the localized treatment
ment of acne infections. This finding confirms that the polyurethane nanofibers loadedof acne infections.
This finding confirms that the polyurethane nanofibers loaded with clindamycin MIP are
with clindamycin MIP are biocompatible with skin tissue, which is not unexpected con
biocompatible with skin tissue, which is not unexpected considering that the formulation’s
sidering that the formulation’s constituents are generally considered safe [39].
constituents are generally considered safe [39].
Figure 5. Clindamycin loaded with MIP polyurethane nanofiber affects HSE-2 cell viability. 100% cell
viability and an untreated cell’s average MTT reduction value correlate. *** Mean p < 0.0015 for ANOVA.
3.8. Determination of Antimicrobial Activity

3.8.1. Antimicrobial Activity of Drug Formulation
The results of the antimicrobial activity evaluation revealed that the Clin-MIP polyurethane
nanofiber has potential activity against all tested strains compared with blank (Clin.) and
control (Gentamycin). Large inhibition zones ranging from 24 mm to 45 mm were deter-
mined by the tested formula against S. epidermidis, S. mutans, and S. aureus. Although
the blank has no activity against the tested Gram-negative strains, the new clindamycin
nanofiber showed good antimicrobial activity against K. pneumoniae, P. vulgaris, and P.
aeruginosa, with inhibition zones larger than those of Gentamycin (Figure 6a,b).
(Clin.) and control (Gentamycin). Large inhibition zones ranging from 24 mm to 45 mm
were determined by the tested formula against S. epidermidis, S. mutans, and S. aureus.
Although the blank has no activity against the tested Gram-negative strains, the new
clindamycin nanofiber showed good antimicrobial activity against K. pneumoniae, P. vul-
Pharmaceutics 2024, 16, 947 garis, and P. aeruginosa, with inhibition zones larger than those of Gentamycin (Figure
21 of 30
6a,b).
(a)
(b)
Figure 6. (a). Antibacterial activity of Clindamycin and Clin-MIP polyurethane nanofibers against
skin profile bacterial strains and (b). Antibacterial activity of Clin. and Clin-MIP polyurethane
nanofibers against S. Epidermis (A), S. mutant (B), S. aureus (C), Pneumonia (D), P. Vulgaris (E), and
P. argues (F). Blank: Clindamycin-Control: Gentamycin. Note: * p = 0.0189, ** p = 0.0015, *** p = 0.0003
and **** p < 0.0001.
3.8.2. Anti-Proliferation Activity of Clin. Nano Formula

The MIC and MBC values confirmed the Clin-MIP polyurethane nanofiber’s higher
activity against the tested organisms than Clin. The Clin-MIP polyurethane nanofiber
has the lowest MICs and MBC values of all tested strains. In contrast, blank (Clin) has
antibacterial activity against two strains, S. aureus and S. mutans, with higher MIC and
MBC values (Table 4). The Clin-MIP polyurethane nanofiber exhibited the highest antibac-
terial activity against the S. aureus strain, with MIC and MBC values of 0.39 µg/mL and
6.25 µg/mL, respectively.
the lowest MICs and MBC values of all tested strains. In contrast, blank (Clin) has anti-
bacterial activity against two strains, S. aureus and S. mutans, with higher MIC and MBC
values (Table 4). The Clin-MIP polyurethane nanofiber exhibited the highest antibacterial
activity against the S. aureus strain, with MIC and MBC values of 0.39 μg/mL and 6.25
Pharmaceutics 2024, 16, 947 μg/mL, respectively. 22 of 30
Table 4. Minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations

(MBC) of clindamycin and Clin-MIP polyurethane nanofiber against the skin profile-tested bacterial
Table 4. Minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC)
strains.
of clindamycin and Clin-MIP polyurethane nanofiber against the skin profile-tested bacterial strains.
Clindamycin (Blank) Clin-MIP Polyurethane Nanofiber
Bacterial Strains Clindamycin (Blank)
MBC Clin-MIP
MIC Polyurethane Nanofiber
MBC
Bacterial Strains MIC (μg/mL)
MBC
(μg/mL) MIC
(μg/mL) MBC
(μg/mL)
MIC (µg/mL)
(µg/mL) (µg/mL) (µg/mL)
Staphylococcus epidermidis (ATCC 12228) 12.5 ± 0.2 12.5 6.25 ± 1.8 6.25
Staphylococcus epidermidis
Staphylococcus (ATCC
aureus (ATCC 12228)
6538) 12.5 ± 3.125
0.2 ± 0.9 12.5
6.25 6.25 ±
0.39 ± 0.21.8 6.25
0.39
Staphylococcus aureus (ATCC 6538) 3.125 ± 0.9 6.25 0.39 ± 0.2 0.39
Streptococcus mutans ATCC 25175 3.125 ± 0.9 12.5 0.78 ± 1.3 3.125
Streptococcus mutans ATCC 25175 3.125 ± 0.9 12.5 0.78 ± 1.3 3.125
Klebsiella pneumoniae(ATCC13883)
Klebsiella pneumoniae (ATCC13883) NA NA NANA 1.56
1.56±±0.90.9 3.9
3.9
Pseudomonas aeruginosa(ATCC90274)
Pseudomonas aeruginosa (ATCC90274) NA NA NANA 6.25
6.25±±0 0 6.25
6.25
Proteus vulgaris
Proteus vulgaris(ATCC13315)
(ATCC13315) NA NA NANA 1.56±±0.45
1.56 0.45 3.9
3.9
NA:no
NA: noactivity.
activity.
3.8.3.
3.8.3. Bacterial
Bacterial Killing
Killing Kinetic
Kinetic Assay
Assay
The
Thebacterial
bacterialkilling
killingkinetics
kineticsof of the
the Clin-MIP
Clin-MIP polyurethane
polyurethanenanofiber againstS.
nanofiberagainst S. aureus
aureus
(ATCC 6538) were assessed and compared with Clin to better investigate its
(ATCC 6538) were assessed and compared with Clin to better investigate its killing capac-killing capacity.
After 180 180
ity. After minminof incubation,
of incubation,Clin-MIP
Clin-MIPpolyurethane
polyurethane nanofiber
nanofiber × MIC)
(1(1× MIC)demonstrated
demonstrated
strong
strongbactericidal
bactericidalactivity,
activity,asasillustrated
illustratedininFigure
Figure7.7.Clindamycin
Clindamycin(1(1× × MIC)
MIC) demonstrated
demonstrated
aa slower
slower killing
killing kinetic
kinetic under
under thethe same
same conditions,
conditions, although
although itit could
could still
still not
not eradicate
eradicate all
all
bacteria after 180 min.
bacteria after 180 min.
Figure 7. Killing kinetics of Clindamycin and Clin-MIP polyurethane nanofiber at 1× MIC of

concentration against S. aureus (ATCC 6538) for different time intervals.
3.9. In Vivo Study

3.9.1. Assessment of Ear Thickness and Anti-Inflammatory Potential
The visual examination of the rat ears in Figure 8 compares the left ear side (untreated
group) and the right ear side treated group; inflammation was evident in all groups re-
ceiving Staphylococcus aureus treatment, as evidenced by a noticeable increase in ear
thickness and redness. As seen in Figure 8, groups II and III experienced a notable de-
crease in inflammation symptoms. Furthermore, it was discovered that group III (treated
with Clin-MIP polyurethane nanofiber) had a very high reduction in inflammation (ear
thickness). In contrast, group II (treated with Clin-MIP) had a high reduction, and group I
(treated with Clin-Suspension) had a low reduction. Preclinical research conducted in a
rat model of acne vulgaris by Tolentino et al., 2021 consistently supported these findings
by demonstrating that clindamycin may have potent antibacterial and anti-inflammatory
targets using polymeric nanocarriers in the event of oily skin disorders; this targeting was
enhanced [7].
Figure 8. Morphological alterations in a rat ear model to identify possible anti-inflammatory.

anti-inflammatory.
3.9.2.The immunomodulatory
Healing effectsPolyurethane
Activities of Clin-MIP of clindamycin were linked
Nanofiber Using theto decreased TNF-α
Viable Count
production and NF-κB
Method in an Animal Modelsuppression. These effects were also seen in a clindamycin derivative
that helped treat painful and inflammatory diseases but had less antibacterial activity; this
The in vivo antimicrobial activity of clindamycin and Clin-MIP polyurethane nano-
is supported by Rodrigues et al., 2023 [41].
fiber significantly decreased the number of S. aureus colonized in infected rat ears compared
Molecular imprinting creates polymer matrix cavities that match the target molecule’s
to the control (untreated model) that was only injected by S. aureus. After completing a treat-
size, shape, and functional groups. Polyurethane nanofibers8 molecularly imprint clin-
ment period, the bacterial counts of S. aureus dropped from 1 × 10 to 39 × 101 CFU/mL and 89
damycin to capture and release it slowly, improving its action at the application site.
× 101 CFU/mL using both Clin-MIP and Clin-MIP polyurethane nanofiber, respectively. In
Adding clindamycin to polyurethane nanofibers allows for continuous medication release.
contrast, the bacterial number of the negative control (untreated) was slightly increased
This delayed-release approach maintains effective clindamycin concentrations at the ap-
to 43 × 108 CFU/mL. In contrast, the positive control treated with clindamycin solution
plication site, prolonging its benefits for acne bacteria and inflammation. Polyurethane
reduced theare
nanofibers bacterial number
molecularly to 92 × 10to
imprinted
2 CFU/mL, as shown in Figure 9.
carry clindamycin to the target spot. Concen-
trating clindamycin at the site of infection and inflammation with molecularly imprinted
polyurethane nanofibers can improve acne treatment while reducing systemic exposure
and side effects.
Clin-MIP Polyurethane nanofiber has a potential effect for treating acne and inflamma-
tion by increasing the stratum corneum penetration of the Clin-MIP Polyurethane nanofiber
combination, notably when integrated into molecularly imprinted polyurethane nanofibers,
mainly inhibits bacterial protein production. It also inhibits peptide bond formation and
bacterial development by binding to the 50S ribosomal subunit of sensitive bacteria such as
Propionibacterium acnes. Clindamycin reduces comedones and inflammatory acne lesions
by lowering the amount of P. acne on the skin. Clin blocks pro-inflammatory cytokines,
including IL-6 and TNF-alpha, which cause acne inflammation. Clindamycin reduces acne
lesion redness, edema, and discomfort by lowering inflammation.
3.9.2. Healing Activities of Clin-MIP Polyurethane Nanofiber Using the Viable Count
Method in an Animal Model
The in vivo antimicrobial activity of clindamycin and Clin-MIP polyurethane nanofiber
significantly decreased the number of S. aureus colonized in infected rat ears compared to
the control (untreated model) that was only injected by S. aureus. After completing a treat-
ment period, the bacterial counts of S. aureus dropped from 1 × 108 to 39 × 101 CFU/mL
and 89 × 101 CFU/mL using both Clin-MIP and Clin-MIP polyurethane nanofiber, respec-
tively. In contrast, the bacterial number of the negative control (untreated) was slightly
increased to 43 × 108 CFU/mL. In contrast, the positive control treated with clindamycin
Figure 9. reduced
solution The total the
number of S. number
bacterial to 92 × 10
aureus (CFU/mL) 2 CFU/mL,
recovered fromas
rats’ ears after
shown treatment
in Figure 9. with
Clin-MIP polyurethane nanofiber. **** p < 0.0001.
to the control (untreated model) that was only injected by S. aureus. After completing a treat-
ment period, the bacterial counts of S. aureus dropped from 1 × 108 to 39 × 101 CFU/mL and 89
× 101 CFU/mL using both Clin-MIP and Clin-MIP polyurethane nanofiber, respectively. In
contrast, the bacterial number of the negative control (untreated) was slightly increased
Pharmaceutics 2024, 16, 947 to 43 × 108 CFU/mL. In contrast, the positive control treated with clindamycin solution 24 of 30
reduced the bacterial number to 92 × 10 CFU/mL, as shown in Figure 9.
2
Figure
Figure 9. The total
9. The total number of S.
number of S. aureus
aureus (CFU/mL) recovered from
(CFU/mL) recovered from rats’
rats’ ears
ears after
after treatment
treatment with
with
Clin-MIP polyurethane nanofiber. **** pp << 0.0001.
nanofiber. ****
3.9.3. Assessment of Inflammatory Biomarkers

Impact of Clin suspension, Clin-MIP, and Clin-MIP polyurethane nanofiber therapy
on the protein profiles of serum inflammatory cytokines
LPS alters the inflammatory cytokines that cause skin inflammation associated with
acne. In earlier research, we found that administering Clin suspension, Clin-MIP, and Clin-
MIP polyurethane nanofibers to inflammation decreased the amounts of NLRP3, TNF-α,
IL-1β, and IL-6 generated by LPS in vitro. Three hours after delivering LPS to rats, we
collected serum samples to examine the impact of Clin suspension, Clin-MIP, and Clin-MIP
polyurethane nanofiber on serum cytokine profiles. Clin-MIP polyurethane nanofiber
therapy dramatically decreased the expression of NLRP3, TNF-α, IL-1β, and IL-6 (40.39%,
79.6%, 70.50, and 73.30%, respectively) in comparison to the LPS group (Figure 10). One
important part of the innate immune system is NLRP3, and pro-inflammatory cytokines
are produced when this protein is activated. Lower levels of NLRP3 suggest that this in-
flammatory pathway is inhibited, which reduces the generation of cytokines like IL-1β and
IL-16. IL-1β, a well-known pro-inflammatory mediator in acute inflammation, indicates
that tumor inflammation may benefit from Clin-MIP polyurethane nanofibers. Numerous
investigations have shown that immunoregulatory cytokines modify the inflammation
caused by wounds. TNF-α regulates IL-6 levels during the initial stages of inflammation.
The alarm-phase cytokine IL-1β has been connected to several symptoms associated with
wounds. Figure 10 shows increased levels of IL-1β receptor antagonists and TNF-α suppres-
sion. Inflammation is a major component of skin pathology. Adipose lipolysis is triggered
by skin tissue leakage and produces toxic unsaturated fatty acids, such as arachidonic acid,
a building block of pro-inflammatory eicosanoids. They produce excessive inflammatory
markers, which can exacerbate illness and cause blisters. Pathogenesis and illness are
encouraged by the innate immune system. Three hours after LPS injection, our research
showed that treating Clin with Clin-MIP polyurethane nanofiber decreased TNF-α, IL-1β,
and IL-6 blood levels. Clin-MIP polyurethane nanofiber may reduce the inflammatory
response to acne skin inflammation by modulating immunoregulatory cytokines [1,42]
Pharmaceutics 2024,
Pharmaceutics 16,16,x 947
2024, FOR PEER REVIEW 27 of 32
25 of 30
Figure10.
Figure The effect
10. The effect of
of Clin
Clin suspension,
suspension,Clin-MIP,
Clin-MIP,and
andClin-MIP
Clin-MIPpolyurethane
polyurethane nanofiber on the
nanofiber on the
inflammatory-related markers NLRP3, TNF-α, IL-1β, and IL-6. Note: * p = 0.0223, *** p =
inflammatory-related markers NLRP3, TNF-α, IL-1β, and IL-6. Note: * p = 0.0223, *** p = 0.0008 0.0008 andand
****pp<<0.0001.
**** 0.0001.

The histological results for the group treated with an alternative formulation on the
rightThe
earhistological
side showedresults for the groupthan
fewer abnormalities treated
the with
left earanside,
alternative
as shown formulation on the
in Table 5 and
right
Figureear11.
side showed
Figure 11a fewer abnormalities
illustrates Group I, the than
leftthe
earleft
sideear(untreated
side, as shown
groupinserving
Table 5asand
Figure 11. Figure 11a illustrates Group I, the left ear side (untreated
control), which exhibited considerable epidermal cell hypertrophy, an apparent increasegroup serving asincon-
trol), whichcell
epidermal exhibited
quantityconsiderable
(hyperplasia), epidermal cell hypertrophy,
and a moderate degree of thickan apparent increase in
stratum corneum
epidermal cell quantity (hyperplasia),
covering (hyperkeratosis). Moderately and thicka coating
moderate degree of
of keratin. The thick stratum
dermal layercorneum
has a
covering
significant(hyperkeratosis). Moderately
rate of inflammatory thick In
cell invasion. coating of keratin.
the dermis, Thesevere
there was dermal layer has a
congestion.
Certain sebaceous
significant glands are destroyed.
rate of inflammatory Clin-Suspension
cell invasion. In the dermis,is used on the
there wasright ear side,
severe and
congestion.
a few mildly
Certain inflammatory
sebaceous glands arecells are in the
destroyed. dermis. There were
Clin-Suspension many
is used hair
on the follicles
right withand
ear side,
asebaceous
few mildly glands in the dermis.
inflammatory cellsMost of the
are in the epidermal
dermis. There layerswere
had amany
normal keratin
hair layer
follicles with
covering them with normal thickness. Most dermal layers showed a normal
sebaceous glands in the dermis. Most of the epidermal layers had a normal keratin layer ordering and
were freethem
covering of congestion.
with normal thickness. Most dermal layers showed a normal ordering and
were free of congestion.
Figure 11b: This depicts Group II, the left ear side (untreated group), where numer-
ous areas of the epidermis are covered in a somewhat thick keratin coating. There is a
thick spot in the epidermis. The dermis showed significant inflammatory cell infiltration,
and the dermal layer showed congestion. The dermis under the skin on the right ear side
(treated with Clin-MIP) shows a small degree of congestion and a tiny infiltration of
mal. The standard thickness was disclosed nearly to the epidermal layer. There was no
hyperplasia noted. A thick coating of keratin covers a few regions of the epidermis.
Figure 11c: demonstrates Group III, the left side (untreated group), where the thick-
ness of the keratin layer has significantly (but not significantly) grown. Significant inflam-
Pharmaceutics 2024, 16, 947
matory cell infiltration exists in the underlying dermis, and the dermis layer showed26sig- of 30
nificant congestion and disarray. There was mild hyperplasia of epidermal cells on the
right side (treated with Clin-MIP polyurethane nanofiber), with certain areas exhibiting
Table 5. Shows the grade of histopathological findings on ear rat skin.
normal epidermal thickness. A slight degree of inflammatory cell infiltration occurs in the
Groups dermis. Numerous
Left F1 hair
Rightfollicles
F1 wereLeftvisible
F2 in several
Right F2places. In Left
the F3
dermis, no conges-
Right F3
tion was observed.
Number of examined fields 10 10 10 10 10 10
Grade
Following treatment with three distinct formulae, the skin’s hyperkeratosis, inflam-
- cell
matory -
+ infiltration,
+ - +
epidermal -hypertrophy,
+ ++ -
and + ++
congestion - improved.
+ -
++ Clin-MIP + was
++
Abnormal changes
Hyperkeratosis administered
5 0 5to Group
6 II
3 and 1 Group
2 5III, respectively.
3 4 4 Compared
2 3 to
5 Group
2 I,7which3 re-0
Inflammatory cells infiltrationceived
2 clin-suspension
6 2 4 treatment,
6 0 1 polyurethane
5 4 2 nanofibers
8 0 recuperated
1 7 2 more4 quickly.
6 0
Thick epidermis (hypertrophy) 6
Compared 1 to 3Group 9 II, Group
1 0 III is
4 better.
3 3
Group 8 III is2the 0most4effective
1 5 9 in1com-
formula 0
Dermal congestion 5 3 2 8 2 0 4 3 3 7 3 0 4 3
parison to other formulas. This implies that, after being used to cure an infection or skin 3 9 1 0
Grade of histopathological findings: - (no abnormality),
disease, the mixture may help the skin layers. + (slight), ++ (mild).
(a)
(b)
Figure 11. Cont.
Pharmaceutics
Pharmaceutics 2024,
2024, 16,
16, x947
FOR PEER REVIEW 29 of 32
27 of 30
(c)
Figure
Figure 11.11. (a)
(a) Displays
Displays photomicrographs
photomicrographs of of skin
skin specimens
specimens from from Group
Group 1. 1. On
On the
the left
left side,
side, the
the
images reveal interrupted dermis, congestion, vacuolated epithelial cells, hyperkeratosis,
images reveal interrupted dermis, congestion, vacuolated epithelial cells, hyperkeratosis, and cellular and cellu-
lar infiltration in the dermis. Increased cellular proliferation of the epidermis and disrupted hair
infiltration in the dermis. Increased cellular proliferation of the epidermis and disrupted hair
follicles are also evident. On the right side, treated with Clin-Suspension, the epidermis and dermis
follicles are also evident. On the right side, treated with Clin-Suspension, the epidermis and dermis
appear intact, with congestion, hyperkeratosis, cellular infiltration, epithelial hyperplasia, and intact
appear intact,
sebaceous withIncongestion,
glands. hyperkeratosis,
(b), the untreated Group IIcellular
exhibitsinfiltration,
disorderedepithelial hyperplasia,vacuolated
dermis, congestion, and intact
sebaceous glands. In (b), the untreated Group II exhibits disordered dermis, congestion,
epithelial cells, hyperkeratosis, and cellular infiltration in the dermis. Similar to Group 1, increased vacuolated
epithelial
cellular cells, hyperkeratosis,
proliferation and cellular
of the epidermis infiltration
and disrupted in follicles
hair the dermis.
are Similar
observed.to Group 1, increased
In contrast, Group
cellular
II treatedproliferation
with Clin-MIP of the epidermis
shows and disrupted
improvement, hairentire
with the follicles arefollicle
hair observed. In contrast,
visible, minimalGroupconges- II
tion, and
treated intact
with sebaceous
Clin-MIP shows glands. (c) showcases
improvement, skinentire
with the specimens from visible,
hair follicle Group III. On the
minimal left side,
congestion,
congestion, vacuolated
and intact sebaceous epithelial
glands. cells, hyperkeratosis,
(c) showcases skin specimens and cellular
from Group infiltration
III. On theinleft
theside,
dermis are ev-
congestion,
ident, along with increased cellular proliferation of the epidermis. However, on the right
vacuolated epithelial cells, hyperkeratosis, and cellular infiltration in the dermis are evident, along side treated
with Clin-MIP-Polyurethane nanofiber, the epidermis and dermis appear healthier, with visible hair
with increased cellular proliferation of the epidermis. However, on the right side treated with
follicles, minimal congestion, and intact sebaceous glands. Note: Epidermis (E), dermis (D), and
Clin-MIP-Polyurethane nanofiber, the epidermis and dermis appear healthier, with visible hair
Hyperkeratosis (K).
follicles, minimal congestion, and intact sebaceous glands. Note: Epidermis (E), dermis (D), and
Hyperkeratosis
Table 5. shows the(K).grade of histopathological findings on ear rat skin.
Groups FigureLeft
11b:F1
This depicts
Right Group
F1 II,Left
the left
F2 ear side (untreated
Right F2 group),
Left F3 where numerous
Right F3
Number of examined fields areas of the epidermis
10 are covered
10 in a somewhat
10 thick
10 keratin coating.
10 There is
10a thick
Grade spot in the epidermis. The dermis showed significant inflammatory cell infiltration, and the
dermal layer- showed
+ - congestion.
+ - + The
- dermis
+ ++under - the+ skin
++ on-the+right
++ ear -side+(treated
++
Abnormal changes
with Clin-MIP) shows a small degree of congestion and a tiny infiltration of inflammatory
Hyperkeratosis 5 0 5 6 3 1 2 5 3 4 4 2 3 5 2 7 3 0
cells. Most hair follicles were connected to sebaceous glands and were normal. The
Inflammatory cells infiltration 2 6 2 4 6 0 1 5 4 2 8 0 1 7 2 4 6 0
standard thickness was disclosed nearly to the epidermal layer. There was no hyperplasia
Thick epidermis (hypertrophy) 6 1 3 9 1 0 4 3 3 8 2 0 4 1 5 9 1 0
noted. A thick coating of keratin covers a few regions of the epidermis.
Dermal congestion 5 3 2 8 2 0 4 3 3 7 3 0 4 3 3 9 1
Figure 11c: demonstrates Group III, the left side (untreated group), where the thickness 0
Grade
of the of histopathological
keratin findings: - (no
layer has significantly abnormality),
(but + (slight),
not significantly) ++ (mild).
grown. Significant inflammatory
cell infiltration exists in the underlying dermis, and the dermis layer showed significant
4. Conclusions
congestion and disarray. There was mild hyperplasia of epidermal cells on the right
side This work
(treated with involved
Clin-MIP synthesizing
polyurethane and characterizing
nanofiber), mixedareas
with certain molecular imprinting
exhibiting normal
polymer-rated into polyurethane
epidermal thickness. nanofiber
A slight degree as a nanocarrier
of inflammatory for topicaloccurs
cell infiltration clindamycin ad-
in the der-
ministration
mis. Numerous to increase clindamycin
hair follicles antibacterial
were visible in several activity.
places. InCombination
the dermis, no therapy was
congestion
thought to be a new tactic for combating bacterial resistance and severe side effects
was observed.
(Graphical
FollowingAbstract). Clin-MIP
treatment polyurethane
with three nanofibersthe
distinct formulae, with homogenous
skin’s morphology
hyperkeratosis, inflam-
and floral
matory forms
cell may be epidermal
infiltration, successfully synthesizedand
hypertrophy, without interpolating
congestion between
improved. different
Clin-MIP was
administered
polymers. to Group
Examined inIIrats’
andears,
Group III, respectively.
earlobes Compared
coated with clin-MIPtopolyurethane
Group I, which received
nanofibers
clin-suspension
revealed treatment,
a 1.3-fold increase polyurethane nanofibers
in permeability recuperated
and little signs ofmore quickly. Compared
inflammation, such as
to Group II, Group III is better. Group III is the most effective formula in comparison to
other formulas. This implies that, after being used to cure an infection or skin disease, the
mixture may help the skin layers.
4. Conclusions
This work involved synthesizing and characterizing mixed molecular imprinting
polymer-rated into polyurethane nanofiber as a nanocarrier for topical clindamycin admin-
istration to increase clindamycin antibacterial activity. Combination therapy was thought
to be a new tactic for combating bacterial resistance and severe side effects (Graphical
Abstract). Clin-MIP polyurethane nanofibers with homogenous morphology and floral
forms may be successfully synthesized without interpolating between different polymers.
Examined in rats’ ears, earlobes coated with clin-MIP polyurethane nanofibers revealed
a 1.3-fold increase in permeability and little signs of inflammation, such as redness and
papules. The study suggests that co-delivering clindamycin together with mixed MIP and
nanofiber may enhance clindamycin effects at the administration site and reduce the risk of
antibiotic resistance emerging.
Author Contributions: S.F.E.: Investigation, writing—original draft, visualization; R.A.: resources,

validation; R.M.E.N.: Investigation Supervision; M.F.M.E.: Supervision, Investigation; A.M.E.H.:
funding acquisition, Investigation; N.A.S.: Investigation; M.S.S.: Investigation, Writing—review and
editing; F.E.H.: Investigation, Writing—review and editing; A.R.: Writing—review and editing, super-
vision, validation; S.L.K.: Investigation, visualization; N.A.E.-N.: Investigation, Writing—review and
editing; A.A.T.: resources, validation; M.E.-N.: Conceptualization, supervision, project administration.
All authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: The Research Ethics Committee-Faculty of Pharmacy, Cairo
University (REC-FOPCU) serial number (code: PI 3218) has approved protocols for animal handling
and experimental procedures. Animal models are among the most important in vivo models for
fundamental parameters such as drug efficiency, safety, and toxicological studies because these pre-
clinical data are necessary before translating into humans. The ARRIVE criteria for animal research
were adhered to in using and handling the study’s animals.
Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.
Data Availability Statement: Data is available on request from the authors.
Conflicts of Interest: The authors declare no conflicts of interest.
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pharmaceutics

Received: 10 May 2024

Pharmaceutics 2024, 16, 947. https://doi.org/10.3390/pharmaceutics16070947 https://www.mdpi.com/journal/pharmaceutics

Keywords: molecularly imprinted polymer; polyurethane nanofiber; acne; antimicrobial; clindamycin;

2. Materials and Methods

2.3.2. In-Vitro Characterization of Clindamycin Molecularly Imprinted Polymer (Clin-MIP)

Fourier Transformer Infrared Spectroscopy (FTIR)

Discussion of Differential Scanning Calorimetry (DSC) Analysis

2.3.3. Fabrication of Composite Polyurethane Nanofiber Dressings by Electrospinning

2.3.4. In-Vitro Characterization of Clin-MIP Polyurethane Nanofiber (CMPN)

Evaluation of Flexibility and Strength

Fourier-Transform Infrared Spectroscopy (FTIR)

Assessment of In-Vitro Drug Release

Drug Release %= AR/AC × 100 (1)

2.4. Ex Vivo Skin Permeation Study

2.5. In Vitro Cell Toxicity Assessment

Cell viability (%) = (sample absorbance)/(control absorbance) ×100 (3)

2.6. Determination of Antimicrobial Activity

2.6.2. Bacterial Proliferation Inhibition Analysis

2.6.3. Bacterial Killing Kinetic Assay

2.7. In-Vivo Study

2.8. In Vivo Evaluation of the Prepared Clin-MIP Polyurethane Nanofiber (CMPN)

2.8.2. Assessment of Inflammatory Biomarkers

2.9. Histopathological Examination

Assessment of Ear Thickness and Anti-Inflammatory Potential on the Skin’s Surface

3. Results and Discussion

3.2. In-Vitro Characterization of Clindamycin Molecularly Imprinted Polymer (Clin-MIP)

MIP 0.0003286 0.1309 0.9994 −3.718 × 10−6 6.42 × 1011 0.9842

Pharmaceutics 2024, 16, x FOR PEER REVIEW 12 of 32

3.2.4. Binding Studies

K2 represents the rate constant for adsorption in a pseudo-second-order reaction. This

3.3. Fabrication of Composite Polyurethane Nanofiber Dressings by Electrospinning

3.4. In-Vitro Characterization of Clin-MIP Polyurethane Nanofiber (CMPN)

Table 2. Physicochemical characteristics of nanofibers (mean ± SD).

Weight Uniformity Thickness Folding Endurance Tensile Strength

3.4.2. Flexibility and Strength Testing Pieces of Nanofiber

3.4.3. Morphology Characterization of Clin-MIP Polyurethane Nanofiber

3.5. In-Vitro Drug Release

Figure 2c shows Polyurethane nanofibers’ FTIR spectra show distinctive absorption

3.5. In-Vitro Drug Release

Figure 3.3. Release

3.6. Ex-Vivo Skin Permeation Study

Table 3. Ex-vivo pharmacokinetic parameters of clindamycin from Clin-MIP and Clin-MIP

Pharmaceutics 2024, 16, 947 3.7. In Vitro Cell Toxicity 20 of 30

3.8. Determination of Antimicrobial Activity

3.8.2. Anti-Proliferation Activity of Clin. Nano Formula

Table 4. Minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations

Figure 7. Killing kinetics of Clindamycin and Clin-MIP polyurethane nanofiber at 1× MIC of

3.9. In Vivo Study

Figure 8. Morphological alterations in a rat ear model to identify possible anti-inflammatory.

3.9.3. Assessment of Inflammatory Biomarkers

3.10. Histopathological Examination

Author Contributions: S.F.E.: Investigation, writing—original draft, visualization; R.A.: resources,

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