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pH-sensitive biocompatible nanoparticles of paclitaxelconjugated poly(styrene-comaleic acid) for anticancer drug delivery in solid tumor of syngeneic mice Manu Dalela, Tulsidas G. Shrivastav, Surender Kharbanda, and Harpal Singh ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.5b07764 • Publication Date (Web): 03 Nov 2015 Downloaded from http://pubs.acs.org on November 7, 2015

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ACS Applied Materials & Interfaces

pH-sensitive biocompatible nanoparticles of paclitaxel-conjugated poly(styrene-comaleic acid) for anticancer drug delivery in solid tumor of syngeneic mice Manu Dalela1, 2, T.G Shrivastav3, Surender Kharbanda4, Harpal Singh1, 2 1

Centre for Biomedical Engineering, Indian Institute of Technology, Hauz Khas, New Delhi-110016, India

2

Biomedical Engineering Unit, All India Institute of Medical Sciences, AIIMS, New Delhi Ansari Nagar, New Delhi-110029, India

3

4

Department of Reproductive Biomedicine, National Institute of Health & Family Welfare, Delhi 110067, India

Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02215

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Abstract In the present study we have synthesized poly(styrene-co-maleic anhydride), a biocompatible copolymer which was further conjugated with paclitaxel (PTX) via ester linkage and self assembled to form poly(styrene-co-maleic acid)-paclitaxel (PSMAC-PTX) nanoparticles. In vitro release of PTX from PSMAC-PTX nanoparticles showed higher release at lower pH than at physiological pH of 7.4, confirming its pH depended release. The cell viability of PSMAC-PTX nanoparticles was evaluated using MTT assay. IC50 values 9.05-18.43 ng/mL of PTX equivalent was observed in various cancer cell lines after 72 h of incubation. Confocal microscopy, western blotting and flow cytometry results further supported that the cellular uptake and apoptosis of cancer cells with PSMAC-PTX nanoparticles. Pharmacokinetic studies revealed that the conjugation of PTX to the PSMAC copolymer not only increase plasma and tumor Cmax of PTX but also prolonged its plasma half life and retention in tumor via EPR effect. Administration of PSMAC-PTX nanoparticles showed significant tumor growth inhibition with improved apoptosis effects in vivo on EAT bearing BALB/c syngeneic mice in comparison with Taxol® without showing any cytotoxicity. On the basis of preliminary results, no subacute toxicity was observed in major organs, tissues and hematological system up to a dose of 60 mg/kg body weight in mice. Therefore, PSMACPTX nanoparticles may be considered as an alternative nanodrug delivery system for delivery of PTX in solid tumor. Keywords: Poly(styrene-co-maleic acid), Paclitaxel, Pharmacokinetics, EAT, BALB/c mice


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1. INTRODUCTION Paclitaxel has been shown to exhibit significant anticancer activity against nonsmall cell lung cancer, head and neck carcinomas and in different solid tumors, including breast, ovarian etc.1.2 Paclitaxel induces the apoptosis in cancer cell by blocking β tubulin subunit of mitotic spindle through mitotic arrest. 3 In cancer chemotherapy, use of paclitaxel is hindered due to its high toxicity, reduced bioavailability, non specificity and poor solubility in aqueous solution. Paclitaxel formulated in Cremophor EL® under the trademark “Taxol®” has been approved by the US Food and Drug Administration to enhance the PTX solubility and to treat patients with various cancers. Severe side effects such as abnormal lipoprotein patterns, hyperlipidemia and anaphylaxis have been observed in Taxol®. Moreover, Cremophor EL® significantly alters the PTX pharmacokinetics and also requires special devices for its administration.4-6 Several methods have been reported to develop new paclitaxel formulations to overcome these limitations of paclitaxel that are devoid of Cremophor EL® as an additive. Polymer based nanodelivery systems have generated keen interest for cancer therapy due to its improved pharmacodynamics and pharmacokinetic profiles of therapeutic drugs. Drug molecules are either released through diffusion from polymer matrix or dissociation of a covalent, ionic or other linkage between drug molecules and polymer matrix. Formulations of PTX in different polymeric nano drug delivery systems have numerous advantages over the standard current chemotherapy. Firstly, the aqueous dispersibility of drug can be greatly increased when the drug is covalently attached to the water soluble polymers or loaded in polymer based systems. Secondly, nanoparticles are small enough in size which enables the efficient and effective delivery of PTX into the tumor site due to endocytosis and enhanced 3 ACS Paragon Plus Environment


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permeability and retention (EPR).7 Conjugation of polysaccharide and polyethylene glycol (PEG) onto the surface of nanoparticles can be used to escape the identification of reticuloendothelial system (RES) in normal cells/tissues and to minimise the side effects of the drugs also. The pharmacokinetic profile of the paclitaxel from the nanoparticles is improved as it increases its half-life and tumor accumulation.8, 9 To deliver PTX by polymer based nano formulations, several approaches have been investigated such as polymeric micelles, water-soluble prodrugs, parental emulsion, complexes with cyclodextrins and polymeric conjugates.10-27 But only a few of these approaches have been approved by the Food and Drug Administration (FDA) probably due to their low drug loading content, batch to batch variations in their bio physicochemical properties, poor biocompatibility, potential toxicity, and instability in vivo. Currently, numerous nanoparticles based paclitaxel formulation are under various phases of clinical trials but only Abraxane have been approved by FDA for breast cancer in 2005 and for non small cell lung cancer (NSCLC) in 2012. 28-30 Abraxane is a biologically active albumin bound PTX nanoparticles with a diameter of ~130 nm. Although Abraxane has shown promising results in cancer patients but still has many limitations like it causes hypersensitivity reactions and hematologic effects to certain patients. Moreover, Abraxane is supplied as a sterile lypophilized powder and each vial has to be reconstituted by through and homogenous mixing with 20 mg/mL of 0.9% sodium chloride injection, improper mixing may result in the formation of foams and clumps. Genexol-PM® is polymeric micelles composed of monomethoxy PEG-block-poly(D, L-lactide) block copolymer (mPEG-b-PDLLA).31 Genexol-PM® was nontoxic at a dose of 300 mg/m2, however, at the maximum tolerated dose (MTD) of 390 mg/m2, a variety of dose limiting toxicities were also noticed, which includes neutropenia and neuropathy. Genexol-PM® is


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currently under Phase IV clinical trial for metastatic breast cancer in US and is being marketed in South Korea and India for treatment of breast cancer and NSCLS. 32, 33 Active drug paclitaxel is released from both Abraxane as well as Genexol-PM® within 3-4 h due to fast degradation of nanoparticles based polymeric carrier. Abraxane have Cmax value of 13µg/mL after 3 h and dropped to 1 µg/mL in 4 h with a single dose of 260 mg/m2 in human patients, while Genexol-PM® shows Cmax of 6.5 µg/mL after 3 h and drop to 1.5 µg/mL in 4 h with a single dose of 390 mg/m2 in human patients. No paclitaxel was detected in the serum after 20 h of drug infusion in both the cases. Zinostatin stimalamer (ZSS) is a potent anticancer drug prepared by conjugation of neocarzinostatin (NCS), with poly(styrene-comaleic acid) and this drug has been approved in Japan since 1994 for primary unresectable hepatocellular carcinoma.34 It has been reported that poly(styrene-co-maleic acid) can form noncovalent association to host’s albumin, which is biocompatible molecule, which may further increase the uptake of anticancer drug by EPR effect.35 In this study, we developed a paclitaxel-conjugated poly(styrene-co-maleic acid) nanoparticles as a representative system for drug delivery in solid tumor. PTX was conjugated to a low molecular weight, nontoxic, biocompatible, amphiphilic diblock copolymer poly(styrene-co-maleic acid) to form PSMAC-PTX nanoparticles. PSMAC-PTX NPs were characterized by particle size, in vitro stability, drug release behaviour and surface morphology. In vitro cytotoxicity of blank nanoparticles (PSMAC) was assessed in various cancer cell lines, while cell killing effects of PSMAC-PTX nanoparticles was tested in different cancer cell lines. PSMAC-PTX nanoparticles was also evaluated for its pharmacodynamics, pharmacokinetic behaviour, toxicity and antitumor efficacy in EAT (Ehrlich Ascites Tumor) bearing syngeneic solid tumor BALB/c mice. We envisage to 5 ACS Paragon Plus Environment


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develop a nanodelivery system with higher drug loading, prolonged and sustained delivery of the paclitaxel using poly(styrene-co-maleic acid) with an aim to reduce toxicity.

2. RESULTS AND DISCUSSION Synthesis and characterization of poly(styrene-co-maleic anhydride) and its conjugation with paclitaxel Synthesis and characterization of poly(styrene-co-maleic anhydride) Poly(styrene-co-maleic anhydride) (PSMA) was synthesized as an alternate copolymer via charge transfer free radical precipitation polymerization at 110 ˚C for 5-6 h using propyl benzene as solvent with high concentration of anhydride groups for drug immobilization (Scheme 1). 1.5% azo bisisobutyronitrile (AIBN) was used in the synthesis to get the desired molecular weight and polydispersity of the synthesized copolymer so that molecular weight of the polymer remained under the renal clearance threshold.36 The obtained white product was further subjected to base catalyzed hydrolysis to get poly(styrene-co-maleic acid) (PSMAC) for immobilization of paclitaxel. In 1H-NMR (Figure. S1) spectrum of PSMA, a broad peak at δ=7.2ppm was observed due to aromatic ring of styrene. Chemical shift appearing at δ=2.5ppm was due to the benzylic proton and the protons present in the maleic anhydride appeared at δ=2.07ppm. Fourier transform infrared (FTIR) spectroscopy was used to study the functional groups of PSMA and PSMAC (Figure. S2). FTIR spectrum of PSMA showed the absorption bands associated with –C=O stretching of anhydride groups of five membered ring at 1783 cm-1 and 1855 cm-1 and C-O-C stretching vibration ( of cyclic anhydride ) at 1218 cm-1. In contrast, a FTIR spectrum of PSMAC (Figure. S2) showed a prominent peak and strong absorption band at ~3500-3800 cm-1 region, corresponding to – OH stretching of carboxylic acid. This shows that anhydride ring was opened up by the


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hydrolysis reaction resulting in generation of free carboxylic acid groups. A broad peak was observed at ~1710-1740 cm-1, corresponds to-C=O stretching of carboxylic acid. In addition to this, a significant decrease in the intensity of peaks at 1778 cm-1 and 1885 cm-1 (-C=O stretching of anhydride groups of five membered ring) was observed, indicating the hydrolysis of anhydride ring. Preparation and characterization of poly(styrene-co-maleic acid) conjugated paclitaxel Poly(styrene-co-maleic acid) conjugated paclitaxel (PSMAC-PTX) was prepared by conjugating paclitaxel with PSMAC in presence of dicyclohexylcarbodiimde (DCC) and 4dimethylaminopyridine (DMAP) as catalyst at room temperature (Scheme 1). Interaction of the hydrophobic conjugated PTX molecules with each other results in collapsing of PSMA chains, to form nanoparticles of 165-185 nm in aqueous solutions as determined by particle size analyzed and scanning electron microscope. The hydrodynamic diameter of the PSMACPTX nanoparticles was found to be 174 ± 10 nm with a polydispersity of 0.134 ± 0.035, and zeta potential of -22.9 ± -1.4 respectively, which is a favourable size for selective tumor uptake in-vivo (Figure. S3).23, 27 In the present study, we found that maleic acid moiety, of PSMAC was conjugated with the paclitaxel via ester linkage as confirmed by FTIR and 1H NMR. The FTIR spectrum of paclitaxel (Figure. S4) showed N-H streching vibrations at 3492-3300 cm-1, CH2 asymmetric and symmetric streching vibrations at 2944-2885 cm-1. The peak observed at 1706 assigned to C=O streching vibration from ester groups. The amide bond was found around 1646 cm-1 and ester bond streching and C-N streching vibrations are observed at 1241 cm-1 and 1276 cm-1 respectively. The FTIR spectra of PSMAC-PTX nanoparticles showed the streching vibration at 1719 cm-1 and C-O streching vibration at 1250 and 1072 cm-1 respectively besides all other characteristics peak of PSMAC and PTX 7 ACS Paragon Plus Environment


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confirming the conjugation of PTX with PSMAC via ester linkage. 1H NMR of PSMAC-PTX nanoparticles (Figure. S5) in D2O showed aromatic components of paclitaxel δ= 7.88-7.38 ppm, and for aliphatic components of paclitaxel δ= 6.27 ppm (C10-H), 5.90 ppm (C13-H), 5.41 ppm and 4.88 ppm (C7-H),4.55 ppm (C20-H), 1.77 ppm (OCOCH3), and 1.00-1.21 ppm (CCH3). PTX content of PSMAC-PTX was quantified and found to be 44.6% (w/w) as determined by high performance liquid chromatography (λmax = 227 nm). Conjugation efficiency of PTX was found to be 78% as calculated by the following formula:

For the clinical use of PSMAC-PTX nanoparticles, stability is an important factor as they could aggregate in physiological fluids and block blood capillaries, and may result in severe problems. PSMAC-PTX nanoparticles stability was performed in normal saline and in Dulbecco’s modified eagle’s medium (DMEM) at 37±2˚C to stimulate the stability of nanoparticles in vitro and in vivo respectively. There was no appreciable increase in particle size as observed by DLS in DMEM and in normal saline, with average particle size remained below 185 nm up to 24 h, demonstrating good and long stability of PSMAC-PTX NPs in aqueous and biological medium as shown in Table S1. Scanning electron micrographs of synthesized PSMAC-PTX NPs are shown in (Figure. 1). We observed that the spherical and rounded morphology of PSMA changed to sugar cube like structure after conjugation with PTX which may be due to the interaction of hydrophobic PTX molecules with each other causing PSMAC chain to collapse to form a sugar cube like nanoparticles of PSMAC-PTX. In our SEM studies, we have randomly picked a field where the morphology of nanoparticles is well defined rather than seeing the average size of the nanoparticles. Further, the 8 ACS Paragon Plus Environment

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conjugation of PSMAC to PTX was determined by electron spray ionization high resolution mass spectrometry (ESI-HDMS) and X-ray photoelectron spectroscopy (XPS). ESI-HD MS analysis of the PSMAC-PTX nanoparticles (Figure. S6) revealed a prominent peak at 1076.72 which corresponds to the conjugation of PSMAC to PTX

calcd

[PSMAC + Paclitaxel =

PSMAC-PTX (220.21 + 853.986 = 1076.196)]. PTX has nitrogen element in its chemical structure while nitrogen is absent in PSMA. Therefore N1s region of the pure PTX was used to determine the presence of drug conjugated with PSMAC. Pure PTX showed the characteristics peak of N1s binding energy 398.763 eV which is also retained after conjugation with PSMAC in PSMAC-PTX [Figure. S7 (a) and(c)]. In vitro PTX release studies Drug release studies from PSMAC-PTX NPs was carried out at 1× PBS buffer solution of pH 7.4 and acetate buffer of pH 5.7 and 4.0, in order to stimulate internal biological environment at 37˚C.37-41 Free PTX (Figure. S8) displayed a fast release, of around 80% of the PTX released into the solution within 6 h of incubation in all pH buffers. In PSMAC-PTX NPs almost negligible initial burst release was observed at pH 7.4 during first 6 h of the study due to the conjugation of PTX with PSMAC by ester linkage. The percent of PTX released from the PSMAC-PTX nanoparticles increased as the pH decreased from 7.4 to 4.2 (Figure. 2). After 24 h, the amount of PTX released in the buffer solutions of pH 7.4, pH 5.7 and pH 4.2 were 3.5 %, 9.7 % and 20.2 % respectively, and those after 48 h were 7.7 %, 14.2 % and 27.1 % respectively. After 3rd day, 41.1 % of PTX was observed at pH 4.2, whereas only minimal amount 11.1% of PTX was released at pH 7.4 and 20.8% was released at pH 5.7. Almost 95 % of PTX was released at pH 4.2, while 51.5% and 25.3% released was observed at pH 5.7 and pH 7.4 on 12th day. The sensitivity of cleavage of ester bonds towards acidic pH can be 9 ACS Paragon Plus Environment


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attributed to the faster release of the PTX from the PSMAC-PTX nanoparticles at pH 4.2. Thus, high release of PTX is due to the sensitivity of ester bond to acidic pH. This effect has been used by various authors for other drug delivery platforms to speed up the drug release inside cancer cells, as in lysosomes pH is 5.0, the concentration of proton is usually 100 folds higher than that of outside the cells where pH is 7.4.38, 42 Thus ester linkage of PSMAC-PTX nanoparticles is more stable at pH 7.4 and prevents PTX release during circulation in blood. In vitro cytotoxicity studies in cancer cell lines Cytotoxicity of PSMAC-PTX NPs was performed in different cancer and normal cell lines at concentration range of 0.01-1.0 mg/mL over 72 h of incubation at 37˚C. Free PTX and PSMAC-PTX nanoparticles are cytotoxic, and both have significant effect on cell viability, for instance the IC50 values of PSMAC-PTX NPs were 101.2, 216.6, 117.4, 97.8, 233.9, 196.6 ng/mL where as IC50 values of free paclitaxel observed were 2.4, 4.1, 2.6, 3.1, 9.4, and 6.6 ng/mL respectively after 48 h of incubation with MCF-7, MDA-MB 231, A549, HeLa, SKHEP1 and HepG2 cancer cells (Figure. 3 and Table S2). The IC50 values for PSMAC-PTX NPs were based on the weight of the PTX in PSMAC-PTX NPs (10 ng of PSMAC-PTX NPs contains 44.6% PTX i.e. 4.6 ng PTX). It should be emphasized that in PSMAC-PTX nanoparticles the cytotoxicity was attributed only to PTX as PSMAC alone does not have any appreciable effect on cell viability. Blank PSMAC nanoparticles (without PTX conjugation) didn’t show any kind of cytotoxicity in different cancer cell lines over 48 h of incubation at the same concentration range (Figure. S9). Higher IC50 values of PSMAC-PTX nanoparticles were observed in various cancer cell lines after 48 h. IC50 values decreased significantly to 9.05-18.4 ng/mL when PSMAC-PTX NPs were incubated with various cancer cell lines for 72 h (Table.S2). Incubation of PSMAC-PTX NPs for longer incubation time achieves more 10 ACS Paragon Plus Environment

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potency due to slow but sustained release of PTX from PSMAC-PTX nanoparticles. Thus, use of the PSMAC-PTX nanoparticles for cancer therapy would be advantageous to avoid the cytotoxicity related with free PTX. In vitro cellular uptake studies using confocal laser scanning microscope (CLSM) To visualize the cellular uptake of nonfluorescent PSMAC-PTX nanoparticles, we adapted a published method43 by introducing red fluorescent Rhodamine B dye loading onto PSMACPTX nanoparticles. Cellular internalization and cellular uptake of Rhodamine B-loaded PSMAC-PTX nanoparticles was examined in MDA-MB 231 cell line after a 2 h incubation using CLSM (Figure 4). To visualize the intracellular localization of PSMAC-PTX NPs, LysoTracker and DAPI was used for colocalization study. A rapid uptake and internalization of the red fluorescence labelled nanoparticles was observed in the cytosol of MDA-MB 231 cells within 2 h (Figure. 4). A higher intracellular concentration of PSMAC-PTX nanoparticles was taken up by cancer cells due to the cellular uptake mechanism via endocytosis of the nanoparticles rather than diffusion of free drug. In this study, we colocalized nanoparticles with lysosomes in order to explore the intercellular behaviour of PSMAC-PTX nanoparticles through EPR effect. Lysosomes were dyed green in the presence of LysoTracker while red fluorescence emitted from Rhodamine loaded PSMAC-PTX nanoparticles further confirmed that nanoparticles were successfully taken up and retained in the cytosol of MDA-MB 231 cells within 2 h of incubation (Figure. 4). These results demonstrated the cellular uptake of PSMAC-PTX NPs by cancer cells.



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Apoptosis assessment studies by flow cytometry and western analysis Flow cytometry experiments were conducted to examined the extent of apoptosis by PSMAC-PTX nanoparticles and free PTX in MDA-MB 231 cells. Cells were double stained with Propidium Iodide (PI) for viability (negative for PI) and Annexin V-FITC for apoptosis (positive for Annexin V-FITC). PSMAC-PTX nanoparticles and free PTX were incubated with MDA-MB 231 cells in serum-containing media at a concentration of 10 ng PTXequivalent of free PTX for 24 and 48 h. PSMAC-PTX nanoparticles resulted in 9.5% early apoptotic cells (positive for Annexin V-FITC only), 5.41% late apoptotic (double positive for Annexin V-FITC and PI) and 6.3 % damaged necrotic cells (Annexin V-FITC negative and PI positive) respectively while free PTX resulted in 12.37 % early apoptotic cells, 9.73 % late apoptotic cells and 10.18 % damaged necrotic cells after 24 h. After 48 h of incubation, PSMAC-PTX nanoparticles resulted in 12.67 % early apoptotic cells, 8.34% late apoptotic cells and 9.62% damaged necrotic cells while free PTX resulted in 15.62 % early apoptotic cells, 12.5 % late apoptotic cells and 14.35 % damaged necrotic cells respectively (Figure. 5). These results indicate that PSMAC-PTX induced apoptotic activity in MDA-MB 231 cells as free PTX but progression is relatively slower in PSMAC-PTX nanoparticles probably due to slow and sustain release of free PTX from the PSMAC-PTX NPs. Importantly negligible apoptosis or cell death was seen in control and void nanoparticles incubated with MDA-MB 231 cell lines. Apoptosis plays a major role in cell cycle and in homeostasis. Caspases, which are critical effectors molecules of cell death process through apoptosis, belongs to a family of cysteine proteases. The activation of caspases is a signaling cascade pathway mediated by proteolysis and Caspase 3 is a key caspase in this process.44-47 The activity of Caspase 3 in apoptosis induction has been detected by a variety of different chemotherapeutic agents.48 12 ACS Paragon Plus Environment

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The expression of Caspases 3 was studied on MDA-MB 231 cells after the treatment with free PTX and PSMAC-PTX nanoparticles for 24 and 48 h (Figure. 6). On incubation with free PTX at 48 h, a sharp and cleaved caspase 3 was observed as higher molecular weight band of 19 kDa and lower molecular weight band of 17 kDa. However, a faint band of cleaved caspase 3 was observed in PSMAC-PTX nanoparticles at 48 h with the corresponding PTX at the same time point. These results suggest that the PSMAC-PTX nanoparticles was successful in initiating apoptosis in MDA-MB 231 cells but the efficacy of action was slow in comparison to free PTX at 48 h. The reason for sharp band at 48 h in case of free PTX is due to increased cell death both by apoptosis and necrosis as confirmed by flow cytometry results (Figure. 7). Hemolysis test Hemolysis and RBC aggregation test were performed to explore the biocompatibility and interaction between the PSMAC-PTX nanoparticles with RBC in buffer at pH 7.4. It was observed that PSMAC-PTX nanoparticles was not hemolytic even up to a concentration of 20 mg/mL while free PTX was highly hemolytic (10%) even at 0.05 mg/mL and reached up to 92.3 % at 1 mg/mL concentration (Figure. 8). No RBC aggregation was monitored by bright field microscopy in PSMAC-PTX nanoparticles while aggregation was clearly visible with free PTX at a concentration of 1 mg/mL (Figure. 9). Results from hemolysis studies thus confirm the biocompatibility of PSMAC-PTX NPs.



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In vivo studies of PSMAC-PTX NPs in EAT solid tumor bearing syngeneic BALB/c mice In vivo tumor growth inhibition studies In vivo antitumor efficacy of PSMAC-PTX NPs was examined in EAT tumor bearing syngeneic BALB/c mice and Taxol® was used as a control. MTD (maximum tolerated dose) of PSMAC-PTX nanoparticles was found to be 80 and 60 mg/kg body weight t while MTD of Taxol® was found to be 20 mg/kg.49 Since, our hypothesis was to show the low toxicity of PSMAC-PTX nanoparticles formulation even at higher concentration compared to Taxol®, and hence dose of nanoparticles was taken 3 × and 4 × higher than that of Taxol®. Most of FDA approved drugs like Genexol–PM and Abraxane which are already in market, uses 3× and 4× dose of PTX in their preliminary studies.50,51 PSMAC-PTX NPs was dispersed in normal saline at a concentration of 22.5 mg/PTX mL (50 mg total weight/mL of PSMACPTX) followed by vortexing for 3-5 min at 500 rpm were given by intravenous injection at 1st, 8th, 15th, and 23rd day at dose of 60 and 80 mg/kg PTX equivalent while a dose of 20 mg/kg Taxol® was given to the groups. In our study dosing was given weekly rather than in a single dose regimen as there are some drawbacks of chemotherapy if given in a single dose regimen such as tumor tissues have limited accessibility of drugs, their unbearable toxicity, side effects and multiple drug resistance development.52 To avoid these issues weekly doses were selected for multi dosing schedule in our experiment as such type of dose regimen were also used by various authors in drug-polymer conjugates studies.53,54 EAT bearing syngeneic BALB/c mice group treated with 60 and 80 mg/kg PSMAC-PTX nanoparticles and 20 mg/kg body weight Taxol® (Figure. 10) showed significant tumor regression as compared to group treated with saline. Tumor volume in mice treated with PSMAC-PTX nanoparticles began to decrease after day 15th post injection, while in Taxol® treated group tumor growth continued 14 ACS Paragon Plus Environment

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until the end of the experiment (50th day). By day 15, tumor volume was 151 mm3 for control group, whereas for 20 mg/kg body weight of Taxol® and 60, 80 mg/kg body weight of PSMAC-PTX nanoparticles tumor volume were 103.6, 85.2 and 72.3 mm3 respectively (P < 0.05). By day 30, the tumor volume was 461.2 for control group, whereas for 20 mg/kg body weight of Taxol® and 60, 80 mg/kg body weight of PSMAC-PTX nanoparticles tumor volume were 125, 76.1 and 60.6

respectively. At the end of the drug regimen dosing

schedule, the group treated with PSMAC-PTX NPs (60 mg/kg body weight) showed significant antitumor activity with tumor growth inhibition of 93.87 % while complete tumor regression was observed with 80 mg/kg body weight of PSMAC-PTX nanoparticles. We have also performed the in vivo tumor inhibition studies with PSMAC-PTX nanoparticles containing 20 mg/kg body weight of PTX at the equivalent dose of Taxol® and observed the tumor inhibition was 71.4% in PSMAC-PTX NPs as compared to 64.5% in Taxol® (P < 0.05). Thus therapeutic efficacy and dispersion stability of PTX has significantly improved after conjugation with PSMAC. Our hypothesis is simply based on the fact that what is the maximum amount of PTX which can be delivered by polymeric system without giving any cytotoxicity, for this reason we have used 3× and 4× dose of PTX in our drug-polymer conjugate. All animals died even with the 30 mg/kg body weight of PTX using Taxol® while all animals survived with 80 mg/kg body weight of PSMAC-PTX NPs dose due to conjugation of PTX with PSMAC. To assess the toxicity of the drug treatment regimen, animal behaviour and change in body weight after drug administration were also examined (Figure 11). It was also observed that mice treated with 20 mg/kg Taxol® became lazy for 1015 min post injection, whereas no behavioural change was noticed after administration of 80 mg/kg body weight of PSMAC-PTX NPs. Toxicity of free paclitaxel and Cremophor EL®



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might be the reason of abnormal animal behaviour of mice treated with Taxol®. In both the treated groups, the weight loss was observed after 1st dose i.e. first week of dosing, but animals start gaining weight after 5th day and attained the original weight after 7th day. During the initial stage of the treatment, a major decline in the bodyweight was observed in mice treated with Taxol® (p lung > kidney > spleen > heart. Compared with Taxol®, the nanoparticles displayed a larger AUC and a longer MRT in plasma and tumor of EAT bearing BALB/c mice, indicating that the PSMAC-PTX NPs were more suitable for higher blood plasma retention and tumor uptake. This phenomenon might be attributed to the fact that tumor tissue have hypervascular permeability and also have impaired lymphatic drainage to facilitate the significant and selective uptake of polymeric nanoparticles ( 10,000 cells counted treatment groups).



Figure 15. Histological images of tumour tissues showing apoptosis using TUNEL assay. Red: apoptosis cells, Blue: DAPI-stained cell nuclei.

Median Survival

100

Percent survival (%)

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