Journal Pre-proof

Mucoadhesive microspheres of ferrous sulphate - a novel approach for

oral iron delivery in treating anemia

Rahul. BS, Lakshmi. S, Sneha Letha S, Sidharth Mohan M,

Rosemary M J

PII: S0927-7765(20)30603-2
DOI: https://doi.org/10.1016/j.colsurfb.2020.111247
Reference: COLSUB 111247

To appear in: Colloids and Surfaces B: Biointerfaces

Received Date: 9 May 2020

Revised Date: 26 June 2020
Accepted Date: 6 July 2020

Please cite this article as: BS R, S L, S SL, M SM, M J R, Mucoadhesive microspheres of

ferrous sulphate - a novel approach for oral iron delivery in treating anemia, Colloids and
Surfaces B: Biointerfaces (2020), doi: https://doi.org/10.1016/j.colsurfb.2020.111247

This is a PDF file of an article that has undergone enhancements after acceptance, such as
the addition of a cover page and metadata, and formatting for readability, but it is not yet the
definitive version of record. This version will undergo additional copyediting, typesetting and
review before it is published in its final form, but we are providing this version to give early
visibility of the article. Please note that, during the production process, errors may be
discovered which could affect the content, and all legal disclaimers that apply to the journal
pertain.

© 2020 Published by Elsevier.

Mucoadhesive microspheres of ferrous sulphate - a novel approach for oral
iron delivery in treating anemia.
Rahul. B.S,a Lakshmi. S,b Sneha Letha S,a Sidharth Mohan. M,a Rosemary M Ja,*
a
Medical Devices Lab, Corporate R & D Centre, HLL Lifecare Ltd., Aakkulam, Sreekariyam. P.O.
Thiruvananthapuram- 695 017, Kerala, India. Tele Phone No. & Fax. (91)471-2774732& (91)471 277 4700.
b
Diagnostics Lab, Corporate R & D Centre, HLL Lifecare Ltd., Aakkulam, Sreekariyam. P.O.
Thiruvananthapuram- 695 017, Kerala, India. Tele Phone No. & Fax. (91)471-2774732 & (91)471 277 4700.
Address: M. J. Rosemary, Scientist E2, Medical Devices Lab, Corporate R & D Centre, HLL Lifecare Ltd.,
Aakkulam, Thiruvananthapuram, Kerala, India.
Email: rosemarymj@lifecarehll.com; mjrose12@gmail.com.

of
Graphical abstract

ro
-p
re
lP
na

Highlights
ur

 Mucoadhesive polymer coated ferrous sulphate microspheres were prepared.

 Mucoadhesion test was conducted to evaluate the microspheres.
 Cell viability studies show that all the microspheres were non-cytotoxic.
Jo

 Cell uptake studies show better uptake to the caco-2 cell lines.
 Animal study show comparative efficacy for the microspheres and ferrous sulphate.

Abstract

Ferrous sulphate is a widely used oral iron supplement with low bioavailability and
substantial side effects. In this study ferrous sulphate has been coated with highly

1
mucoadhesive polymers such as hydroxypropylmethyl cellulose (HP), chitosan (CS) and
carbopol (CP) by spray drying technique to produce mucoadhesive polymer coated
microspheres with good yield and high encapsulation efficiency. Mucoadhesive coating may
allow these microspheres to get attached to the intestine and hence better absorption of
ferrous sulphate may be achieved. In vitro release studies from the microspheres show that
the release follows non-Fickian zero order drug release. CP and CSHP coated microspheres
showed good swelling(~1200 to 2400 %) and mucoadhesion properties (58 to 95 %)
indicating that they can swell and get attached to the intestine for longer time period as
compared to free ferrous sulphate. All the microspheres were found to be non-cytotoxic in
Caco2 cell lines and fibroblast cell lines. Cell uptake studies conducted on Caco2 cell lines

of
showed that uptake of microspheres containing ferrous sulphate has an increased and
sustained release to the cell as compared to free ferrous sulphate. Though cell uptake studies

ro
showed an increase in uptake for ferrous sulphate microspheres, comparable efficacy was
observed upon administering ferrous sulphate microspheres and ferrous sulphate to phenyl
hydrazine induced anemic rats.
-p
Key words: Mucoadhesion, anaemia, ferrous sulphate, oral delivery, efficacy studies
re
1. Introduction

As per world health organization (WHO); anemia is a serious global health problem affecting
lP

more than two billion people worldwide [1]. Iron deficiency is the common cause of anemia
[2, 3]. Recently iron deficiency was ranked by WHO as seventh in a list of top ten global
preventable risks for death, disease and disability [4, 5]. WHO data shows that the frequency
na

of iron deficiency in developing countries is 2-5 times greater than that of developed
countries due to various factors such as infections and malnutrition. Failure to include iron-
ur

rich foods in the diet and inappropriate dietary intake along with variation in bioavailability
due to presence of absorption inhibitors in the diet are some of the important factors
responsible for iron deficiency [6].
Jo

An approximate estimate of prevalence of anaemia among various categories of world-wide

population is as follows: preschool age children (47%), pregnant women (41%), non-
pregnant women (30%), school-age children (25%) and people older than 60 years of age
(24%) [7]. Increased iron need during growth and pregnancy and chronic blood loss
contribute to higher prevalence of anaemia among women and adolescent girls. Maternal

2
anaemia is often associated with poor intrauterine growth, increased risks of preterm birth
and higher perinatal morbidity and mortality rates [8, 9].
Oral iron supplementation, especially ferrous sulphate is cheap, safe and effective in
correcting iron deficiency. [10,11]. However consumption of ferrous sulphate gives rise to
side effects such as stomach pain, nausea, vomiting, constipation, diarrhea and epigastric pain
and most of these side effects are attributed to the fraction of iron that remains unabsorbed
following digestion and remains localised in the intestinal lumen [12], [13], [14], [15]. High
discontinuation rates in oral therapy is often associated with above mentioned side effects and
unpleasant taste.
The key to make patient compliant iron formulations is to reduce the amount of unabsorbed

of
iron from the gut and thus the idea to coat ferrous sulphate with a mucoadhesive polymer is
proposed. Mucoadhesive coating by gastrointestinal targeting polymers will increase the

ro
residence time of the ferrous sulphate microspheres in the duodenum and will eventually help
in increased iron absorption from duodenum and proximal jejunum [16, 17]. Mucoadhesive
microspheres can exhibit prolonged residence time at the site of application and facilitate an
-p
intimate contact with the underlying absorption surface thus contributing to improved
absorption and therapeutic performance of a drug [18, 19].
re
Here the polymers such as hydroxypropyl methyl cellulose (HP), carbopol (CP) and chitosan
(CS) were chosen to coat ferrous sulphate. These polymers were chosen due to their excellent
lP

mucoadhesive property and applications in gastro intestinal targeted drug delivery [20], [21],
[22]. Coating of the ferrous sulphate by different polymers was previously investigated using
spray drying [23], [24], [25]. Although the above mentioned polymers have been utilized for
na

coating various other drugs, this is the first time they are being attempted to coat ferrous
sulphate aimed at improving oral iron supplementation. The polymer and drug ratios were
optimized to get higher encapsulation efficiencies and controlled drug release. In vitro drug
ur

release of the microspheres were done at pH 1.2 and pH 7.4 and kinetic modeling of the same
was carried out in order to study the drug release. % mucoadhesion and swelling studies were
Jo

performed for all the samples to check the feasibility of mucoadhesion. The cytotoxicity
studies were done on fibroblast and Caco 2 cells and cell uptake studies were conducted on
well characterized human Caco 2 cell lines. Efficacy studies were conducted on phenyl
hydrazine induced anemic rat models.
2. Experimental

2.1 Materials and methods

3
Ferrous sulfate (dried) was purchased from Himedia Laboratories Pvt. Ltd (India). Carbopol-
974P NF was obtained as a gift sample from Arihant Innochem Pvt. Ltd (India).
Hydroxypropyl methylcellulose (HPMC E15) was obtained as a gift sample from Colorcon
Asia Pvt. Ltd (India). Chitosan was procured from India Sea food, Cochin (India). Ascorbic
acid and ethanol was procured from Merck (India).
Dulbecco’s modified eagle medium (DMEM) for the cell culture studies was purchased from
Lonza, Switzerland. Penicillin and streptomycin, was purchased from Thermo Fisher (USA).
Fetal bovine serum was purchased from Gibco (USA). PBS was purchased from Himedia
(India). Trypsin, acridine orange and ethidium bromide were procured from Sigma Aldrich
(USA). All other reagents used were of analytical grade.

of
2.2 Preparation of microspheres

ro
2.2.1 Preparation of spray solution
The method of preparation of spray solution depends on the type of polymer used. CP (1%
w/v) was dissolved in distilled water to form a homogenous polymer solution at room
-p
temperature. HP (1% w/v) was prepared by dissolving HP in 10% ethanol water mixture.
Encapsulation efficiency of the CS microspheres was found to be very low; hence a polymer
re
combination of CS and HP (CSHP) was prepared instead of CS alone. CS (85% Degree of
acetylation) and HP in various ratios were dissolved in 1% aqueous acetic acid solution and
lP

stirred overnight for complete dissolution.

Ferrous sulphate (1% w/v) along with ascorbic acid (15:1) was dissolved in water at room
temperature and mixed thoroughly with the prepared polymer solution and homogenized for
na

30 minutes at 10000 RPM. Ascorbic acid was added along with ferrous sulphate to enhance
the absorption of ferrous sulphate. In the case of CS microspheres, gluteraldehyde was added
ur

as the crosslinking agent.

2.2.2 Spray drying

Jo

Ferrous sulphate incorporated microspheres were prepared by spray drying technique. All
samples were spray dried using “Jay instruments SPRAY MATE” laboratory spray dryer
with a standard 0.5 mm nozzle. During spray drying process; the feed solutions were sprayed
at an inlet temperature of 135 – 140 ₀ C and an outlet air temperature of 70-80 ₀ C with a flow
rate of 2 mL/minute. The dry microspheres were collected from the bottom of the cyclone

4
separator and weighed to determine the process yield. Twelve microspheres were designed
and prepared by varying the drug-polymer ratio - 1:1, 1:2, 1:3 and 1:4 (w/w).

2.3 Scanning electron microscopic (SEM) studies

Surface morphology of the prepared microspheres was examined using Zeiss EVO 18 and
HITACHI S2400 scanning electron microscope. The samples were coated with gold to ensure
conductivity.
2.4 Estimation of iron by Atomic Absorption Spectroscopy (AAS)
The iron content of the microspheres was determined using AAS by GBC 932 plus atomic
absorption spectrophotometer.
2.5 Estimation of encapsulation efficiency

of
Encapsulation efficiency is defined as the ratio of the amount of drug encapsulated to that of
the drug used initially for microsphere preparation. It was found out by the method given in

ro
the supplementary information.
2.6 In-vitro release
-p
Approximately 20 mg of the spray dried microspheres were weighed and dispersed in 200
mL of pH 1.2 HCl solution and pH 7.4 PBS to study the release. In vitro release was found
re
out by the method given in supplementary information. All the experiments were carried out
in triplicates at 37 ₀ C.
2.7 Release Kinetics
lP

In order to evaluate the release kinetics from the microspheres, the release data was fitted on
various release models: Zero order (cumulative % drug release vs time), first order (log
cumulative % drug remaining vs time), Higuchi (cumulative % drug release vs square root of
na

time), Hixson-Crowell (log cumulative % drug released vs log time) and Korsmeyer–Peppas
(log cumulative percentage of drug release vs log time) models at two different pH conditions
ur

1.2 and 7.4 [26]. The release rate constants were found out from the slope of the plot and
correlation coefficient (r2) close to unity was taken as the order of release.
Jo

If the release exponent “n” value is 0.45 or less, the release mechanism follows “Fickian
diffusion” and higher values of n from 0.45 to 0.89 for mass transfer follows a non-Fickian
model (anomalous transport). The drug release follows Higuchi model of drug release and
case II transport if the n value is 0.89. For the values of n higher than 0.89, the mechanism of
drug release is regarded as super case II transport [27].
2.8 Ex-Vivo mucoadhesion test conducted on isolated sheep’s intestine

5
The mucoadhesive property of the microspheres was evaluated by ex-vivo wash off test
[28].The details of the test are given in the supplementary information.
2.9 Swelling studies
Swelling studies were conducted following the method given in the supplementary
information.
3.0 Cell culture studies
Fibroblast cells and Caco 2 cells were procured from National Centre for Cell Science, Pune,
India. The cells were sub-cultured and maintained in Dulbecco’s modified Eagle’s medium
(DMEM) supplemented with 10 % and 20 % fetal bovine serum (FBS) respectively in a CO2
incubator at 37 ₀ C, 5% CO2 and saturated humidity.

of
3.1 Cell cytotoxicity by MTT assay
Cell viability assay was performed following the protocol given by Mossman et al., 1974 [29]

ro
(supplementary information).

Morphological assessment of apoptosis by fluorescent imaging

-p
Briefly, cells were seeded in 96-well plates at a seeding density of 5 x 103 cells and then
treated with different concentrations of microspheres for 48 hours. After washing once with
re
phosphate buffered saline (PBS), the cells were stained with 100 μL of a mixture (1:1) of
acridine orange-ethidium bromide (4 μg/mL) solution. The cells were immediately washed
lP

with PBS and observed under fluorescence microscope (Leica-DFC 295) at 450-490 nm.

3.2 Uptake studies

Caco-2 cells were seeded onto 6-well plates at a seeding density of 3 x 104 cells/cm2 in CO2
na

incubator at 37 ₀ C. The medium used was DMEM supplemented with 20% FBS and 6 µmL-1
glutamine. The media was replaced every two days. Iron supplementation was provided
according to the methodology cited by Zariwala et al., 2013 [30]. On the 13th day, cell
ur

monolayers were incubated in serum free minimum essential media (MEM) for 24 hours after
washing three times with wash solution (140 mM NaCl, 5 mM KCl, 10 mM PIPES buffer,
Jo

pH 6.7, 37 ₀ C). On day 14, the treatment media was prepared by adding 10, 20 and 40 µg/mL
of final iron concentrations of microspheres (CP, HP and CSHP) and ferrous sulphate (F10,
F20 & F40) to aliquots of test media (MEM, pH 5.8, buffered with 2-[N-Morpholino] ethane-
sulfonic acid 10 mM). Caco-2 cell monolayers were washed three times with wash solution
and iron enriched treatment media was added to cell monolayers. Plates were incubated for 2
and 4 hours at 37 ₀ C on a rotating plate incubator (25 RPM). Test media was then aspirated
and cells were washed twice with wash solution and once with surface bound iron removal

6
solution (wash solution plus 5 µM sodium hydrosulphite and 1 µM bathophenanthroline
disulfonate) [30, 31]. Cells were placed in an incubator for 24 hours, then lysed using lysis
buffer (50 mM NaOH supplemented with 1 µg/mL protease inhibitor cocktail) and harvested
by scraping the cells and collecting the lysate into micro centrifuge tubes.
Iron absorption analysis
Iron absorption was determined by measuring total ferritin protein concentration in cell
lysates using a spectrophotometric ELISA kit at 620 nm (Ramco laboratories INC- Cat No:
S-22) using instructors manual, as described previously [32]. Details are given in the
supplementary information.
3.3 Efficacy studies

of
Male Wistar rats (5-6 weeks age) purchased from Hylasco Biotechnology Pvt. Ltd, India
were used for the study. Rats were allowed to acclimatize to the experimental room

ro
conditions for a period of five days. Animals were housed in an environmentally monitored
air-conditioned room with adequate fresh air supply (>20 air changes per hour), maintained at
a temperature of 22± 3 ₀C and humidity of 30 - 70 %, with 12 hour light and 12 hour dark
-p
cycle. The temperature and relative humidity were recorded once daily. They were housed in
a polypropylene cage, 3 animals per cage, with clean sterilized corn cob as the bedding
re
material. They were provided with standard rodent autoclaved diet (Altromin Spezialfutter
Gmbh & Co. KG, Cat No. 1324) and water ad libitum during the experimental period.
lP

Veterinary examination of animals were performed during the study period of acclimatization
and recorded. Sick/moribund, injured and animals which showed abnormal behavior were
excluded from the study. The tests were performed as per quality management systems 17025
na

and in compliance with principles of Good Laboratory Practice (GLP). Animals were
randomized into different treatment groups based on body weight. The animals were formed
into 6 groups, 6 rats in each to perform the study. Ferrous sulphate microspheres and raw
ur

powder formulations were administered once daily via oral gavage for a period of 14 days.
The clinically recommended dose of different ferrous sulphate formulations is 300 mg/day.
Jo

Based on the USFDA guidelines for dose conversion between human and rat species, the rat
equivalent dose for ferrous sulphate is calculated as 31 mg/kg. As per literature reports on the
rat model of phenylhydrazine induced anemia, ferrous sulphate was found to be effective
when tested at a dose of 50 mg/kg. Based on the clinical dose and reported literature, ferrous
sulphate powder and microspheres were administered at a dose of 50 mg/kg. Blood sampling
procedures were performed in compliance with standard protocol using isoflurane anesthesia
and blood was withdrawn via retro orbital plexus using capillaries into micro centrifuge vials
7
containing EDTA. Hematological parameters were measured on day 4 of treatment period
(interim measurement) and on the day of termination of the study (day 14). Animals were
subjected to blood sampling for assessment of hematology parameters such as hemoglobin
(Hb), red blood cells (RBC) and packed cell volume (PCV) using an automated hematology
analyzer (ADVIA2120,Siemens Ltd., India). Animals were sacrificed at the end of treatment
period by following a standard protocol using CO2 euthanasia. The grouping of animals,
details of induction of anemia and evaluation gastric tolerance are given in the supplementary
information.

3.4 Statistical analysis

All the results were carried out in triplicate. Results are expressed as mean ± SD. One‑ way

of
Student’s test was used to determine statistical significance. Differences were considered to
be significant for values of P < 0.05.

ro
3.0 Results and Discussion

-p
Mucoadhesive polymer (CP, HP and CSHP) coated ferrous sulphate microspheres were
developed by spray drying technique. Based on the preliminary experiments, spray drying
re
conditions such as inlet temperature, compressed air flow and liquid flow rate were set at
150₀ C, 1.2 m3/min and 2 mL/min, respectively.
lP

Please insert Table 1.

na

Spray dried particles were collected in an amber colored bottle and stored at room

temperature. All the microspheres prepared were subjected to gamma (γ) sterilization. The

microspheres were γ irradiated at 25 ₀ C, 60 % relative humidity in dark condition using 60Co

ur

gamma cell at an irradiation dose of 25 kGy (Microtol Sterilisation Services Pvt. Ltd.
Bengaluru, India). SEM analysis, encapsulation efficiency and in vitro release study was
Jo

performed on the microspheres to confirm that properties are retained after γ irradiation
(Data not shown).
The different composition of the polymers, amount of ferrous sulphate, amount of ascorbic
acid, product yield and the encapsulation efficiencies of all the prepared microspheres are
given in the Table 1.The yield of the spray drying process is an important indicator for the
economic feasibility and scalability of the process [33]. It was reflective from the several

8
studies that the process variables such as drug to polymer ratio, inlet temperature and pump
flow rate significantly affects the size and yield of the microspheres [34], [35]. From Table 1,
it is clear that for CP microspheres, as the polymer amount is increased; the yield of the
product also increased from 63 to 77 %, for HP yield was reduced from 69 to 55 % and for
CSHP the yield varied from 38 % to 49 %. Encapsulation efficiencies of the CP microspheres
increased from 44 to 108 %, whereas for HP microspheres as the polymer amount was
increased; encapsulation efficiency varied from 90 to 80 %. For CSHP microspheres the
encapsulation efficiency was found to be low for CSHP1 and CSHP 3 (59 % & 58 %) while it
was higher for CSHP2 and CSHP4 (89 and 84%). Hence it can be inferred that ferrous
sulphate and the different polymers in the ratio of 1:2 shows good encapsulation efficiency

of
and yield.

ro
Please insert Figure 1.

SEM images of the empty microspheres as well as ferrous sulphate loaded microspheres are
-p
given in the Figure 1. The particle size of CP empty microspheres and HP empty
microspheres are in the range of 6 µm and 3.3 µm respectively. After loading ferrous
re
sulphate, CP and HP particle size changed to 8.4 µm and 4.7 µm respectively with a few
particles in the size range of 10 and 20 µm in case of HP. In case of HP microspheres, large
lP

number of polymer particles were observed along with spherical particles while with CSHP
microspheres, both empty and drug loaded microspheres showed spherical shaped
microspheres with a size of 10 µm.
na

Supplementary information Figure 1 & 2 shows the DSC and TGA of all the microspheres.
DSC thermograms of ferrous sulphate shows an endotherm at 300 ₀ C. But the DSC
thermograms of CP, HP and CSHP microspheres does not show the endotherm indicating
ur

that the drug has been completely incorporated within the polymer and hence the peak of the
drug has been vanished [36]. TGA graphs of the samples are given as supplementary
Jo

information. It was observed that degradation starts at 200 ₀ C, 250 ₀ C and 300 ₀ C for CP,
CSHP and HP polymers respectively. All the microspheres showed degradation at around
200 ₀ C.

Please insert Figure 2 and Table 2

9
 Figure 2 shows the % cumulative release of the ferrous sulphate at two different pH of 7.4 for 8
hours and 1.2 for 120 minutes. We may see that in the acidic pH CP, HP and CSHP
microspheres are showing 68 to 88 %, 81 to 96 % and 69 to 83 % of release after two hours. In
the case of pH 7.4, all the four types of CP, HP and CSHP samples released drug in the range
of 73 to 100 % after 7 hours.
The in vitro dissolution data for pH 1.2 & 7.4 was fitted with different release mechanisms
such as zero order, first order, Higuchi, Korsemeyer-Peppas and Hixson-Crowell [36, 37].
Upon analyzing Table 2, it can be observed that all the microspheres follow zero order kinetics
and Korsemeyer-Peppas model release kinetics as they showed maximum linearity
(supplementary information). For zero-order kinetics, the release of an active agent is only a

of
function of time and the process takes place at a constant rate independent of active agent
concentration [36]. Here we can see that the ‘n’value is more than 0.89 and hence the release

ro
can be characterized as non Fickian case II and super case II transport and follows zero order
kinetics[37], [38].

-p
It can be seen that the pH of mouth is in the range of 6.2 to 7.6 and as per Figure 2 ; the release
of all ferrous sulphate microspheres were very less in the first hour of the release at pH 7.4,
re
hence it can be assumed that these microspheres will not produce any bad taste in the mouth
[39]. % mucoadhesion and swelling index of the microspheres were also studied [40].
lP

Please insert Figure 3

The swelling index of the 3 types of microspheres: CP, HP and CSHP microspheres are shown
in the Figure 3. From Figure 3A, we can see that CP microspheres showed a swelling % of
na

1200. HP1 and HP2 showed a swelling % of 1600 whereas HP3 and HP4 started disintegrating
after sometime. CSHP1 and CSHP2 microspheres showed swelling % of 500 whereas CSHP3
ur

and CSHP4 showed a swelling % of 2400. Along with swelling index; mucoadhesion is another
important property which can affect the absorption of ferrous sulfate by the body. It was found
Jo

that all the microspheres especially CSHP and CP showed better mucoadhesion as compared to
HP microspheres. CP, HP and CSHP microspheres showed a mucoadhesion % of 58-75 , 20 and
75-95 respectively. It can be observed that, out of the three types of polymer used; CP and
CSHP coated ferrous sulphate microspheres showed the best swelling and mucoadhesion
properties.

Please insert Figure 4

10
Figure 4C and 4D shows the % viability of the fibroblasts and Caco-2 cell lines when incubated
with 12 different microspheres. It can be seen that the microspheres are nontoxic to the cell lines
but in turn had a proliferating effect on both the cell lines. As the dose increased from 250
µg/mL to 500 µg/mL, a dose dependent decrease in the cell viability can be observed. It was
observed that the microspheres exhibited more than 80 % cell viability at 250 µg/mL and 500
µg/mL except for HP2 at 500 µg/mL on Caco-2 cells. Most of the microspheres were observed
to be possessing proliferating effect (>100) at 250 µg/mL on fibroblast cell lines. CP, HP and
CSHP empty microspheres showed almost 100 % viability in the cell lines, implying that the
polymers used for coating iron showed a nullified effect in cell cytotoxic studies. Higher

of
concentrations of 750 and 1000 µg/mL were found to be comparatively toxic (data not shown).
The acridine orange/ethidium bromide stain is a viability stain that differentiates between viable

ro
and apoptotic cells. Ethidium bromide is a DNA intercalating class of compound that can pass
through the membrane of a dead or dying cell and stains nuclei orange fluorescence whereas
acridine orange is a membrane-permeable dye that will stain all cells in the sample with green
-p
fluorescence. The present treatment showed the viable and apoptotic/necrotic cells formed
followed by 48 hours of treatment with the microspheres (Figure 4. A & B). Almost all the
re
nuclei of cells appeared to possess green fluorescence which showed that the cells were viable
and the microspheres were non toxic at 250 µg/mL.
lP

Please insert Figure 5

na

Caco-2 cell lines produce ferritin based on the amount of iron (ferrous sulphate) present in the
cell environment. Hence the amount of ferritin formed can be used to quantify the absorption
of ferrous sulphate into cells [41]. Here different amounts of ferrous sulphate (F1, F2 & F4)
ur

and the three different microspheres with a concentration of 10 µg/mL, 20 µg/mL and 40
µg/mL were added to Caco-2 cells and incubated for 2 and 4 hours. The uptake of ferrous
Jo

sulphate and the amount of ferritin ng /mg of protein was checked after 2 and 4 hours. As
shown in the Figure. 5, after 2 hours; lower concentrations of microspheres (HP2-10, HP2-20;
CSHP2-10, CSHP2-20; CP2-10, CP2-20) showed an increase in mean concentration of ferritin
(ng) /mg of protein in the range of 50 to 162 ng /mg in comparison with ferrous sulphate (F10
& F20). But in the case of higher concentrations (HP2-40, CSHP2-40, CP2-40 vs F40) the
increase was only in the range of 2 to 17 ng /mg of protein for the microspheres. After 4 hours
of the study; there was an increase in the range of 30 to 152 ng /mg of ferritin in the lower

11
concentrations in comparison with ferrous sulphate. But in the case of higher concentrations
the increase was only in the range of 15 to 33 ng /mg of ferritin. It can be observed that in
lower concentrations; there was a significant increase in absorption for ferrous sulphate
microspheres, whereas at higher concentration it was not significant as compared to ferrous
sulphate. Upon comparing the amount of ferritin; there was significant increase in lower
concentrations of microspheres and ferrous sulphate after 2 and 4 hours, while at higher
concentrations it was not significant. This showed that for ferrous sulphate incorporated
microspheres; controlled release of iron into the cells in the lower concentrations takes place. It
can also be observed that at higher concentrations amount of iron released was the same after 2
and 4 hours for ferrous sulphate as well as microspheres. There are reports of increased gastro

of
intestinal uptake of drugs by the process of bio adhesion for solid lipid coated nanoparticles
[42].

ro
A six month accelerated stability analysis (40 ₀ C, 75 % RH) was conducted on CP2, HP2 and
CSHP2 microspheres as per ICH guidelines to check the stability of the microspheres.
-p
Microspheres in the ratio of 1:2 were chosen for the stability study as they were having good
encapsulation efficiency values along with other properties. It was found that CP2
re
microspheres were retaining all the important properties after the stability analysis and hence
passed the stability analysis (data not shown). Thus CP2 was chosen for conducting the
efficacy study. The efficacy study was conducted on phenyl hydrazine induced anemic Wister
lP

rats as per the protocol given in the experimental section using CP2 microspheres (Figure. 6).
All experimental procedures in the study were approved by the Institutional Animal Ethics
Committee (IAEC, CPCSEA registration No. 1192/PO/RcBt/S/08/CPCSEA of Anthem
na

Biosciences) and in accordance with the CPCSEA guidelines for animal experimentation.

Please insert Figure 6

ur

Hemoglobin levels (Hb) (g/dL): Treatment with Ferrous sulphate microspheres resulted in a
statistically significant (p<0.0001) elevation in hemoglobin levels on day 14 (13.1 g/dL vs 10.7
Jo

g/dL) compared to phenylhydrazine control. Red blood cells (106 cell/µL): Ferrous sulphate
microsphere treatment showed a statistically significant increase in red blood cells (5.2×106
cells/µL vs 4.1×106 cells/µL) compared to phenyl hydrazine control. Packed cell volume
(PCV, %): Treatment with ferrous sulphate microspheres resulted in a statistically significant
(p<0.0001) increase in PCV (40.3 % vs 31.1 %) on day 14. Treatment with the ferrous sulphate
also resulted in significant (p<0.0001) increase in hematological parameters. The Hb, RBC and
PCV were returned to normal levels after 2 weeks of treatment with ferrous sulphate and CP 2

12
microspheres compared to the phenyl hydrazine control. Normal animals (Group 5 and 6)
treated with ferrous sulphate and microspheres did not show any hematological changes
compared to the vehicle control. There was no effect on the weight of the animals during the
study with no adverse clinical signs. When you compare the Hb, RBC and PCV after 4 and 14
days of the study of treatment with ferrous sulphate and the CP 2 microspheres; we can see that
they are comparable. The gastric tolerance was also checked and on gross examination
followed by histopathology evaluation, no changes were observed in gastric mucosa of animals
treated with ferrous sulphate and CP 2 microspheres.

4.0 Conclusions

The study reported the development of highly mucoadhesive CP, HP and CSHP coated ferrous

of
sulphate microspheres. Prepared microspheres were characterized using scanning electron
microscope and it showed micropores on the surface of the microspheres. Microspheres with

ro
drug polymer ratio 1:2 showed good encapsulation efficiency and yield. Further
characterizations were performed using DSC and TGA analysis. In vitro release studies
-p
conducted on all the microspheres showed that the release follows non Fickian, zero order
kinetics. All the microspheres showed good cell viability on fibroblast as well as Caco-2 cell
re
lines. Cell uptake studies conducted on Caco-2 cell lines showed that iron microspheres are
showing slow and increased release into the Caco2 cells as compared to free ferrous sulphate
lP

in the lower concentrations. CP and CSHP formulations showed best mucoadhesion properties.
CP2 formulation was found to be stable in the stability analysis conducted. Efficacy studies
conducted on CP2 microspheres showed improvement in blood parameters as compared to the
na

phenyl hydrazine control rats; but comparable efficacy when compared to ferrous sulphate
administered rats with no gastric irritation. Hence it can be concluded that mucoadhesive
microspheres of ferrous sulphate having good encapsulation efficiency, yield, swelling
ur

capacity, mucoadhesion capacity, increased cell uptake in caco2 cell lines and efficacy at pat
par with ferrous sulphate are potential candidates for treating anemia.
Jo

Author Credit

Rahul.B.S: Experiments carried out, made the protocols for development and characterisation
and part of manuscript preparation.

Lakshmi .S: Conducted the cell culture experiments and part of manuscript preparation.

13
Sneha Latha. S: Planned the animal experiments and part of manuscript preparation.

Sidharth Mohan M: Prepared samples for the animal studies and part of the experimental team.

Rosemary M J: Conceptualization of the idea, planning the experiment

along with other team members, writing the article, correction. Ideas

6.0Funding
The author(s) disclosed receipt of the following financial support for the research, authorship,
and/or publication of this article: this work was fully supported by Science and Engineering

of
Board (SERB), Department of Science and Technology, Govt. of India, under the study
number EMR/2016/002298.

ro
7.0 Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal
relationship that could have appeared to influence the work reported in this paper.
Acknowledgements
-p
RMJ is grateful to Science and Engineering Board (SERB), Department of Science and
re
Technology, Govt. of India for financial support. Authors thank HLL Lifecare Ltd. for
providing all the required facilities for the execution of the project.
lP

References
na

[1] World Health Organization. http://www.who.int/nutrition/topics/ ida/en/.

[2] A. P. Singh, J. Siddiqui, L. L. Diosady, Food Bioproc Tech:Characterizing the pH-
ur

Dependent Release Kinetics of Food-Grade Spray Drying Encapsulated Iron Microcapsules

for Food Fortification 11 (2) (2017) 435-446.
[3] A. Louis, J. R. Kazal, Am Fam Physician: Prevention of Iron Deficiency in Infants and
Jo

Toddlers, Oct 1 66 (7) (2002) 1217-1224.

[4] M. G. Zariwala, N. Elsaid, T. L. Jackson, F. Corral-Lopez, S. Farnaud, S. Somavarapu, D.
Renshaw, Int. J. Pharm: A novel approach to oral iron delivery using ferrous sulphate loaded
solid lipid nanoparticles 456 (2) (2013) 400-407.

[5] R. F. Hurrell, S. Lynch, T. Bothwell, H. Cori, R. Glahn, E. Hertrampf, Z. Kratky, D.

Miller, M. Rodenstein, H. Streekstra, B. Teucher, E. Turner, C. K. Yeung, M. B.

14
Zimmermann., Int J Vitam Nutr Res: Enhancing the absorption of fortification iron 74 (6)
(2014) 387–401.

[6]C. S. Gautam, L. Saha, K. Sekhri,Medscape J Med:Iron Deficiency in Pregnancy and the

Rationality of Iron Supplements Prescribed During Pregnancy 10 (12) (2008) 283.

[7]S. G. López, P. Herrero, F. Bermejo, C. Villa, K. Aspuru,

Int J Gen Med: Optimal management of iron deficiency anemia due to poor dietary intake (4)
(2011) 741-750.

[8] W. K. Simmons, J. D. Cook, K. C. Bingham, M. Thomas, J. Jackson, M. Jackson, N.

Ahluwalia, S. G. Kahn, A. W. Patterson, Am. J. Clin. Nutr: Evaluation of a gastric gastric
delivery system for iron supplementation in pregnancy 58 (5) (1993) 622-626.

of
[9] K. Kalaivani ,Indian J Med Res: Prevalence & consequences of anaemia in pregnancy
(130) (2009) 627-633

ro
[10] N. C. Andrews, N. Engl. J. Med.: Disorders of iron metabolism (341) (1999) 1986–1995.
-p
[11] W. H. Crosby, Arch. Intern. Med.: The rationale for treating iron deficiency anemia 144
(3) (1984) 471–472.
re
[12] N. Martnez-Navarrete, M.M. Camacho, J. Martnez-Lahuerta, J. Martnez-Monzo, P. Fito,
Food Res. Int.: Iron deficiency and iron fortified foods – a review. 35 (2) (2002) 225–231.

[13] K. Schümann, T. Ettle, B. Szegner, B. Elsenhans, N. W. Solomons, J. Trace Elem. Med.

lP

Biol:On risks and benefits of iron supplementation recommendations for iron intake revisited
21 (3) (2007) 147–168.
na

[14] P. P. Yu, Y. Z. Chang, P. Yu, J Pharma Care Health Sys: A more Effective Iron
Supplement for Sports Anemia and Anemia of Inflammation (S4) (2015).
ur

[15] L. Saha, P. Pandhi, S. Gopalan, S. Malhotra, P. K. Saha, MedGenMed: Comparison of

efficacy, tolerability, and cost of iron polymaltose complex with ferrous sulphate in the
treatment of iron deficiency anemia in pregnant women 9 (1) (2007).
Jo

[16] A. Sosnik, J. das Neves, B. Sarmento, Prog. Polym. Sci.: Mucoadhesive polymers in the
design of nano-drug delivery systems for administration by non-parenteral routes: A review
39 (12) (2014) 2030-2075.

[17] M. E. Conard, J. N. Umbreit, Am. J. Hematol.: Iron absorption and transport-An update
64 (4) (2000) 287-298.

15
[18] C. Prego, M. Garcia, D. Torres, M. J. Alonso, J Control Release: Transmucosal
macromolecular drug delivery 101 (1-3) (2005) 151-162.

[19] V.K. Yadav, A. B. Gupta, R. Kumar, J. S. Yadav, B. Kumar, J Chem Pharm Res:
Mucoadhesive Polymers: Means of Improving the Mucoadhesive Properties of Drug Delivery
System 2 (5) (2010) 418-432.

[20] J. d. Neves,. B.Sarmento, Mucosal Delivery of Biopharmaceuticals: Biology, Challenges

and Strategies, Springer, 2014, NewYork.

[21] L. Kumar, S. Verma, B. Vaidya, V. Gupta, Bioadhesive Polymers for Targeted Drug
Delivery:Nanotechnology-Based Approaches for Targeting and Delivery of Drugs and
Genes, Elsevier, 2017, London.

of
[22] A. K. Nayak, R. Maji, B. Das, Asian J Pharm Clin Res: Gastroretentive drug delivery
systems: a review 3 (1) (2010) 2-10.

ro
[23] V. Dueik, L. L. Diosady,J Food Process Eng: Microencapsulation of iron in a reversed
enteric coating using spray drying technology for double fortification of salt with iodine and
iron 40 (2) (2016).
-p
[24] A. Sosnik, K. P. Seremeta, Adv Colloid Interface Sci: Advantages and challenges of the
spray-drying technology for the production of pure drug particles and drug-loaded polymeric
re
carriers 223 (2015) 40-54.

[25] D. Santos, A. C. Maurício, V. Sencadas, J. D. Santos, M. H. Fernandes, P. S. Gomes,

lP

Biomaterials - Physics and Chemistry - New Edition: Spray Drying: An Overview (2018).

[26] P. Costa, J. M. Sousa Lobo, Eur. J. Pharm. Sci.: Modeling and comparison of dissolution
profiles 13 (2) (2001) 123-133.
na

[27] J. Ansary, A. K. Chaurasiya, K. B. Huq, Asian J. Med. Biol. Res.: Formulation and
evaluation of metformin HCl floating microspheres 1(3) (2016) 396.
ur

[28] C. M. Lehr, J. A. Bouwstra, A. M. Schacht, H. E. Junginger, Int J Pharm: In vitro

evaluation of mucoadhesive properties of chitosan and some other natural polymers (78)
(1992) 43-48.
Jo

[29]B. T. Mossman, M. J. Gray, L. Silberman, R. Lipson,Obstetrics and Gynecology (1974).

Identification of neoplastic versus normal cells in human cervical cell culture(43) (1974)
635–639.

[30] Zariwala MG, Elsaid N, Jackson TL, Corral López F, Farnaud S, Somavarapu S,
Renshaw D. A novel approach to oral iron delivery using ferrous sulphate loaded solid lipid
nanoparticles. Int J Pharm. 456 (2) (2013) 400-7.

16
[31] Glahn RP, Gangloff MB, Van Campen DR, Miller DD, Wien EM, Norvell WA.
Bathophenanthrolene disulfonic acid and sodium dithionite effectively remove surface-bound
iron from Caco-2 cell monolayers. J Nutr.125(7) (1995) 1833-40.
[32] G. K. Braga, W. P. Oliveira, Dry Technol: Manufacturing Drug Loaded Chitosan
Microspheres by Spray Drying: Development, Characterization, and Potential Use in
Dentistry 25 (2) (2007) 303-310.

[33] D. E. Oakley,Chem Eng Prog: Produce uniform particles by spray-drying 93 (10) (1997)
48-54.

[34] J. Adamiec, Z. Modrzejewska, Dry Technol: Some Structural Properties of Spray-Dried

Chitosan Microgranules 23 (8) (2005) 1601-1611.

of
[35] M. S. Islam, M. Moniruzzaman, R. Rony, T. Haque, S. J. Pharm. Sci.: Effects of coating
on the release profile of drug combination from hydrophilic matrix pellets 2 (2) (2009) 53-58.

ro
[36] K. Mladenovska, O. Cruaud, P. Richomme, E. Belamie, R. S. Raicki, M. C. Venier-
Julienne, E. Popovski, J. P. Benoit, K. Goracinova, Int. J. Pharma: 5-ASA loaded chitosan–
-p
Ca–alginate microparticles: Preparation and physicochemical characterization 345 (1-2)
(2007) 59-69.
re
[37] N. A. Peppas, Pharm Acta Helv: Analysis of Fickian and non-Fickian drug release from
polymers 60 (4) (1985) 110-111.
lP

[38] R. S. Harland, A. Gazzaniga, M. E. Sangalli, P. Colombo, N. A. Peppas, Pharm. Res.:

Drug/Polymer Matrix Swelling and Dissolution 5 (8) (1988) 488-494.
[39] W. A. Ritschel, G. A. Thompson, Methods Find Exp Clin Pharmacol : Monitoring of
drug concentrations in saliva: a non-invasive pharmacokinetic procedure 5 (8) (1983) 511-
na

525.
[40] J. Jacob, E. Mathiowitz, D. Enscore, M. Schestopol, Bioadhesive drug delivery system
with enhanced gastric retention, W.O. Patent # 2003051304 A2 (2003).
ur

[41] P. Sharp, Int. J. Vitam. Nutr. Res. Methods and options for estimating iron and zinc
bioavailability using Caco-2 cell models: benefits and limitations 75 (6) (2005) 413–421.
[42] P.Severino, T.Andreani, A.S.Macedo, J.F.Fangueiro, M.H. Santana, E.B.Souto, J Drug
Jo

Deliv. 24(2011) doi:10.1155/2012/750891.

9. List of referees

name university area email

17
1. Prof. Gang Rice university nanomedicine gang.bao@rice.edu
Bao

2. Prof. Eliza National univeristy Cancer cells bieflse@nus.edu.sg

fong of singapore

3. Prof. Rita Toronto univeristy Soft tissues rita.kandel@sinaihealthsystem.ca

Kandel

A B

of
ro
-p
re
C D
lP
na
ur

E F
Jo

18
 Figure. 1 SEM image of ferrous sulphate microspheres. A & B are the images of empty
microspheres of CP and drug loaded CP2 microspheres. C & D are the images of empty
microspheres of HP and drug loaded HP 2 microspheres, E & F are the images of empty
microspheres of CSHP and drug loaded CSHP 2 microspheres.

cp1
100 cp2 A
cp3

of
cp4
HP1
% Cumulative Release

HP2
80 HP3
HP4
CSHP1

ro
CSHP2
CSHP3
60 CSHP4

40

20
-p
re
20 40 60 80 100 120
Time (Min)

120
CP1
CP2
B
CP3
lP

CP4
HP1
100 HP2
% Cumulative Release

HP3
HP4
CSHP1
80 CSHP2
CSHP3
CSHP4
na

60

40

20
ur

0
0 1 2 3 4 5 6 7 8 9
Time (Hrs)
Jo

Figure.2 Cumulative in vitro release profiles of the ferrous sulfate in the PH 1 (A) and PH (B).
7.4.

19
 2000
2000
1800
A
0.5Hr
1Hr
2Hr
B 0.5Hr
1Hr
2Hr
1600 3Hr 3Hr
4Hr 1600 4Hr

of
1400 5Hr 5Hr
6Hr
6Hr
7Hr
% Swelling

1200
% Swelling

7Hr
1200
1000

800
800

ro
600

400 400
200

0 0
cp1 cp2 cp3 cp4 cp (em) hp1 hp2 hp3 hp4

3000

C
0.5Hr
1Hr
2Hr
Time Interval

100
D
-p Time Interval

percentage mucoadhesion
percentage mucoadhesion (%)

2500 3Hr
4Hr
5Hr 80
re
2000 6Hr
7Hr
% Swelling

60
1500

1000 40
lP

500 20

0
0
cshp1 cshp2 cshp3 cshp4 cshp (em) CP1 CP2 CP3 CP4 HP1 HP2 HP3 HP4 CSHP1CSHP2CSHP3CSHP4
Time Interval formulations
na

Figure.3A, B & C show the % swelling of CP, HP and CSHP microspheres and 5D shows
the percentage mucoadhesion of CP, HP and CSHP microspheres.
ur
Jo

20
 of
A 160

140
C CSHP 250
500
2

ro
120

100
% Viability

Control CP2
80
to
-p
60

40
re
20

0
HP2 CSHP2 CP1 CP2 CP3 CP4 HP1 HP2 HP3 HP4 CSHP1 CSHP2 CSHP3 CSHP4

22
lP

160 HP4
B 250
140 D 500

120
Cont
na

100
Control
rol CP2
% Viability

80

60
ur

40

20
Jo

HP2 CSHP2 0
CP1 CP2 CP3 CP4 HP1 HP2 HP3 HP4 CSHP1 CSHP2 CSHP3 CSHP4

Figure. 4Acridine orange-ethidium bromide dual staining in (A) fibroblast and (B) Caco-2
cells 48 hours post treatment with the different microspheres. C & D shows the % viability of
all the microspheres in (C) fibroblast and (D) Caco-2 cells.

21
 700
2 hours

of
600 4 hours
Ferritin uptake, ng/mg of protein

*** ***
ns
ns ***
500

ro
400

300

200
-p
re
100

0
lP

F-10 F-20 F-40 HP2-10 HP2-20 HP2-40 CSHP2-10 CSHP2-20 CSHP2-40 CS2-10 CS2-20 CS2-40

Figure.5 Concentration of ferritin levels (ng/mL) in Caco-2 cell lines after treating with
different microspheres and ferrous sulphate after 2hr and 4 hr.
na
ur

18 10
50
Normal control
Phenyl hydrazine control
PHZ+FeSO4 Raw powder-50mg/kg
A 16 C 9
E
Red blood cells (106cells/µL)

40 PHZ+FeSO4,Microspheres-50mg/kg 14 8
Packed cell volume (%)

Feso4 Rawpowder-50mg/kg
Hemoglobin (g/dL)

Feso4 Microspheres-50mg/kg 7
12
Jo

30 6
10
5
8
20 4
6
3
4
10 2
2 1

0 0 0
5 6 5 6 5 6
Day 4 Day 4 Day 4
10
60 20
9
18
B F
Red blood cells (106 cells/µL)

50 8
16
D
Packed cell volume (%)

7
14
Hemoglobin (g/dL)

40
6
12
5
30 10

8 4

20 3
6

4 2
10
2 1 22
0 0 0
5 6 5 6 5 6

Day 14 Day 14 Day 14

Figure. 6 Effect on the hematological parameters of carbopol coated ferrous sulphate
microspheres after 4th& 14th day of the efficacy study. (A) & (B) shows thepacked
cellvolume, (C) & (D) shows the hemoglobin level, (E) & (F) shows the effect on red blood
cells.

code Amount of polymer Amount of Amount of Process Encapsulation

(mg) FeSO4 a.a (mg) Yield (%) Efficiency (%)
(mg)
CP1 1000 1000 60 63.1 44.72
CP2 2000 1000 60 67.9 99.72
CP3 3000 1000 60 75.6 109.15
CP4 4000 1000 60 77 108.22
HP1 1000 1000 60 68.9 93.6

of
HP2 2000 1000 60 61.1 88.05
HP3 3000 1000 60 46.5 80.83
HP4 4000 1000 60 55.3 90.27

ro
CSHP1 500 (CS) + 500 (HP) 1000 60 38.3 59.72
CSHP2 500(CS) + 1500 (HP) 1000 60 53.5 89.16
CSHP3 1500 (CS) +500 (HP) 1000 60 42.8 58.88
CSHP4 2500(CS) + 500 (HP) 1000 -p
60

Table 1: Amount of the polymer, drug & ascorbic acid, process yield and encapsulation
49.2 84.44
re
efficiency values of the different ferrous sulphate microspheres.

Zero order Korsmeyer-Peppas

lP

PH code R2 K0 n R2 Intercept

CP1 0.9998 0.7285 .9939 1 0.117

CP2 1 0.5596 .9842 1 0.2176
CP3 0.9998 0.529 .9662 1 0.2107
na

CP4 0.9998 0.5713 1.0209 1 0.2986

HP1 1 0.7751 .9867 1 0.1057

1 HP2 0.9999 0.716 .981 0.9998 0.099

HP3 0.9999 0.7878 .9679 0.9999 0.0293
ur

HP4 0.9996 0.6721 .9776 0.9996 0.1181

CSHP1 .9999 .52 .8154 0.9993 1.5925
CSHP2 .9999 .6042 .9836 0.9999 1.5694
CSHP3 1 .6813 .9807 1 1.6253
Jo

CSHP4 1 .6481 1.0169 1 1.5826

CP1 0.9992 11.38 .9817 0.9988 1.0976
CP2 0.9997 11.39 .9957 0.9999 1.0691
CP3 0.999 14.341 1.2286 0.998 .9706
CP4 1 14.022 1.089 0.9999 1.0852
7.4 11.14 .9284 0.9972 1.178
HP1 0.9993
HP2 0.9969 9.0637 .8526 0.9912 1.1229
HP3 0.9783 10.000 .9187 0.9964 1.1812
HP4 0.9903 10.957 .9043 0.9919 1.195
CSHP1 .9996 2.5448 .9272 0.9997 1.0898
CSHP2 .9615 5 .9533 0.9994 1.0876

23
 CSHP3 .9656 54.698 .9082 0.9994 1.2674
15.13 .9841 0.9991 1.1287
CSHP4 .9829
Table. 2 Various calculated parameters and coefficients of zero order and Korsmeyer-Peppas
release models for pH 1 and pH 7.4.

of
ro
-p
re
lP
na
ur
Jo

24