IN VITRO

1) Co-delivery of docetaxel and doxorubicin using biodegradable PEG-PLA micelles for

treatment of breast cancer with synergistic anti-tumour effects
R.K.Dey & Prajna Mishra. 2018 chose methoxy – poly (ethylene glycol) -Poly (lactic acid) [mPEG-
PLA]. mPEG-PLA is a boon because of its specificity, increase in permeability, biodegradability and
its capacity to enclose both hydrophilic and hydrophobic drugs. In this work they used MCF-7 cell
lines. The copolymer was encapsulated with docetaxel (DTX) and doxorubicin(DOX). DOX drug is
inserted between base pairs of DNA/RNA causing changes in its structure and in turn leading to its
arrest. DTX impede the microtubule disassembly and blocks the G2/M phase of cell cycle. These
drugs are more effective when given in combination. To transport across the membrane the drugs
enters the cells via endocytosis. Endosomal escape occurs and the nano formulation releases the drugs
from the endosome. Now apoptosis / inhibition of cell growth occurs. Cytotoxicity on MCF-7 cells is
nil when blank PEG-PLA is used. Which shows the biocompatibility of PEG-PLA NPs with MCF-7
cell lines. After 48h of exposure the percentage of viable cells when MPEG-PLA-DOX-DTX was
used were less (33%) when compared to just free delivery of DOX-DTX (43%). So, these drugs
efficacy is more effective when they are co delivered on MCF-7 cell lines. 98% of the drugs were
released in the time frame of 96 h at pH 7.4.

2) Preparation of anastrozole loaded PEG-PLAnanoparticles: evaluation of apoptotic response

of breast cancer cell lines

PLA-PEG-PLA NPs were chosen in this study as they are non-toxic and biodegradable. Anastrozole (ANS) (2,
2-[5-(1H-1, 2, 4-triazol-1-ylmethyl)-1.3-phenylene]), which is commercially available as Arimidex, is an
aromatase inhibitor (AI). Anastrozole (ANS) acts by reversibly binding to aromatase and inhibiting its actions,
thus inhibiting the conversion of androgens to estrogen. MCF-7 cell lines were used in this experiment. The
release profile of three different batches of ANS-NPs was measured at different time intervals over a period of
144 hours. empty PEG-PLA NPs showed no significant cytotoxicity against MCF-7 cells (inhibition ,10%). On
the other hand, ANS-NPs inhibited the growth of MCF-7 cells after incubation for 72 hours. drug-release profile
of ANS-NP showed EE OF 67%, sustained release over 144 hours and did not exhibit burst effect in the first
few hours. This prolonged release can be explained by the binding affinity between the polymer and the drug,
and the capacity of the polymer to incorporate the drug. ANS-NPs presented successful delivery of the drug,
which was confirmed by in vitro cytotoxicity studies and by analyzing the differences in the expression of
MAPK3, c-MYC, and MCL-1 in MCF-7 cells treated with free ANS and with ANS-NPs.

3) A Novel Combined Micellar System of Lapatinib and Paclitaxel with Enhanced

Antineoplastic Effect Against Human Epidermal Growth Factor Receptor-2 Positive
Breast Tumor In Vitro

A combined PEG–PLA micelles of LPT and PTX (PPM-LP) were prepared to enhance anticancer
effect against HER-2-positive breast cancer. Paclitaxel (PTX), a taxanes widely used for
chemotherapy. Overexpression of HER-2 results in higher resistance against chemotherapeutic agents
such as PTX. Overexpression of the HER-2 can lead to the increased tyrosine–kinase activity.
Lapatinib (LPT), a tyrosine kinase inhibitor that targets EGFR and HER-2,8 not only effectively
inhibit the proliferation of HER-2 overexpressing breast cancer but also strongly sensitize originally.
PTX-resistant tumor to PTX in vitro and in vivo. Combining LPT and PTX into one formulation
confers great advantages over the separate formulations. Human breast cancer cells SKBr-3 and
MDA-MB-231were used. However, for HER-2-positive SKBr-3 cells, IC50 value of Taxol, PPM–
PTX, and PPM-LP was 1.362 } 0.240, 1.499 } 0.547, and 0.701 }
0.315 :g/mL; IC50 value of PPM-LP was significantly lower than that of PPM–LPT (p < 0.05), Taxol
(p < 0.05), and PPM– PTX (p < 0.05), which indicated adding LPT could obviously
induce synergetic effect with PTX against the HER-2-positive cells. PPM–LPT showed
no difference with control, indicating it singly did not cause early apoptosis, but the combined
micelles of PPM-LP showed a statistically important increase of apoptosis as compared with
PPM–PTX, 51.9 } 1.1% versus 42.2 } 0.8% (p < 0.05, 9.7% increase). After treated with PPM-LP,
more cells were arrested in G2/M and S phase. Cytotoxicity test indicated that LPT could obviously
sensitize SKBr-3 (HER-2 positive) to PTX; whereas for MDA-MB-231 (HER2
negative), LPT showed no effect on chemosensitization of PTX. Combined LPT and PTX into
nanosized micelles showed the strongest antineoplastic effect as compared with control, PPM–LPT,
and PPM–PTX.

4) Co-delivery of Sildenafil (Viagra®) and Crizotinib for Synergistic and Improved Anti-
tumoral Therapy

(Crizotinib and Sildenafil loaded PEG-PLA micelles) was used here. Crizotinib, has the ability to
induce apoptosis via Caspase-3, as well an anti-angiogenic potential (23). Furthermore, it is also
described that this drug specifically inhibits the MDR1 efflux transporter, coded by ABCB1 gene.
Sildenafil, a potent phosphodiesterase inhibitor, also possess an antagonist effect on ABC efflux
pumps, since it inhibits the action of MDR1, breast cancer resisting protein
(BCRP) and multidrug resistance-associated protein 1 (MRP1), which are present in breast cancer
cells. (MCF-7) breast carcinoma cells were used. anti-tumoral effect of Crizotinib was markedly
pronounced with only 22% of MCF-7 remaining viable after its administration. The simultaneous
incubation of cells with Crizotinib and Sildenafil resulted in an abrupt decrease in cellular viability
(only 10% of cells remained viable), when compared with the incubation of Crizotinib alone. after
only 24 h the PL C micelles decreased breast cancer cells viability up to 37% .More importantly, the
administration of dualloaded micelles reduced cell viability to 25%, after 24 h incubation. After 48 h,
differences in cell viability in the single and dual micelle formulations were still observed. At this
time, single-loaded PL C micelles promoted a decrease inMCF-7 cell viability levels up to 14%.
This evidences the role of Crizotinib in promoting cell death trough apoptosis, that was also reported
emphasizing that this is a valuable approach to promote breast cancer cell death. when both drugs are
delivered through PEG-PLA micellar carriers the therapeutic outcome is enhanced, since a 2.7 fold
reduction in MCF-7 cell viability was obtained, when Crizotinib and Sildenafil were simultaneous
delivered by these DDS and with only half of the dose administered in free drug formulations.

5) Dodecanol-poly(d,l-lactic acid)-b-poly (ethylene glycol)-folate (Dol-PLA PEG-FA)

nanoparticles: Evaluation of cell cytotoxicity and selecting capability in vitro

The Dol-PLA-PEG-FA polymer were used. The selection of FA as a ligand is because FA receptor
has been widely proved to be over-expressed in many human cancers, FA has high binding
affinity (Kd ≈ 10−10 M) to its receptor, low immunogenicity and good storage stability. To improve
the stability of NPs, highly hydrophilic H2NPEG- NH2 was chemically added to form a PEG layer on
the NP surfaces which generates repulsive force between NPs so as to prevent flocculation. The cell
proliferation and cytotoxicity of Dol-PLA-PEG-FA NPs were evaluated by means of CCK-8 assay
using MCF-7 and EC as model cells and normal fibroblast cells (CCL-110). at the same incubation
time, the uptake of the NPs for MCF-7 was significantly higher than that for CCL-110, suggesting
much higher affinity and selecting capability of the NPs to MCF-7. The results from
the CCK-8 assay revealed that the obtained NPs2 are non-toxic to either tumor cells MCF-7 or normal
cells EC. the cellular uptake of FA in physiological environments is mainly controlled by receptor-
mediated endocytosis. The more FA receptor in cell membrane means more uptake
of FA into cells. And the cell uptake experiment of Rhodamine 6G-loaded Dol-PLA-PEG-FA NPs
demonstrated
that the existence of folate on the NPs surfaces significantly increased the selecting capability of the
NPs to the tumor cells MCF- 7 compared to the normal fibroblast cells CCL-110, suggesting a
receptor-mediated endocytosis pathway of Dol-PLA-PEG-FA NPs. Dol-PLA-PEG-FA polymer and
its nanoparticles could be used as a safe drug carrier with strong
tumor-specific targeting capability for tumor chemotherapy.

6) LATER Nanoparticles loaded with ferrocenyl tamoxifen derivatives for breast cancer
treatment

two organometallic triphenylethylene compounds (Fc-diOH and DFO), with strong antiproliferative activity in breast cancer
cells, but insoluble in biological fluids, were incorporated in two types of stealth nanoparticles (NP): PEG/PLA nanospheres
(NS) and nanocapsules
(NC). MCF-7 cell lines were used here. Fc-diOH and DFO were incorporated efficiently in NC and NS. Compared to
metal coordination
complexes, organometallic compounds should be more stable,
because their metal–ligand bond has a stronger covalent character. Compound Fc-diOH
(1,1-di(4_-hydroxyphenyl)-2-ferrocenylbut-1-ene, Fig. 1) is an
analog of OH-Tam, where the tamoxifen _- aromatic benzyl
ring has been replaced by the aromatic ferrocene moiety, and
the amino side-chain has been replaced by a second hydroxyl
group. It is our most efficacious cytotoxic
compound to date. In compound DFO (1,2-di(4_-hydroxyphenyl)-
1-[4__-(2__-ferrocenyl-2__-oxoethoxy)phenyl]but-1-ene,
the amino side-chain of OH-Tam has been changed into a
carbonylferrocene moiety. DFO has
shown a weaker antiproliferative activity towards cancer cells
but good affinity for the estrogen
receptors. thatDFOhas a better antiestrogenic
activity than compound Fc-diOH. Precisely, its luciferase inhibition
activity is ten times stronger than that of Fc-diOH. bothDFO and Fc-diOH -loadedNCsand NSs have a weaker
transcription inhibition
activity than free DFO and Fc-diOH.

7) Biological Characterization of Folate-Decorated Biodegradable

Polymer−Platinum(II) Complex Micelles
In this paper two amphiphilic block copolymers: FA-PEG-b-PLA and mPEG-b- P(LA-co-MCC/Pt) was used.
The drug molecules were wrapped in the core part of the micelles, while FA resided in the corona of the
micelles to effectively play a role of targeting moiety. A folic acid-conjugated copolymer, folic acid
poly(ethylene glycol)-block-poly(L-lactide) (FA−PEG-PLA), with similar chemical structure was
chosen for targeting. Folic acid (FA) receptors are overexpressed in several human tumors including
ovarian and breast cancers and rarely expressed in normal tissues, therefore, folic acid has been
chosen as the targeting moiety. Platinum(II) drugs cause apoptosis of cancer cells via chelating with
the DNA strands to form Pt−DNA adducts. In vitro evaluation was performed by using MCF-7
cancer cells. The cells were incubated for 0.5 h with oxaliplatin, M(Pt) and FA-M(Pt) at an equivalent
initial Pt concentration of 0.25 μM, the Pt contents in the cells were 616.4, 487.8 and 745.4 ng of
Pt/(106 cells) for MCF-7 cells and 1148.6, 1057.6, 1338.4 ng of Pt/(106 cells). Therefore, the
effectiveness order is FA-M(Pt) > M(Pt) > oxaliplatin. IC50 Values of Oxaliplatin, M(Pt) and FA-
M(Pt) against MCF-7 cell lines are 2.2, 6.9, 3.9 respectively. Biodistribution studies showed that
targeted micelles FA-M(Pt) led to increase in the Pt content in the tumor site and consequently
displayed a substantially better antitumor efficacy.