CHAPTER-4 RESULT AND DISCUSSION

Transcription

1 CHAPTER-4 RESULT AND DISCUSSION 4.1. Synthesis of PLGA-gemcitabine conjugate PLGA gemcitabine conjugate was synthesized using zero-length crosslinkers, DCC, and NHS. It is the most common activation method of carboxylic group, and the formed active esters are suitable for coupling with primary amines (Veronese and Morpurgo, 1999). The method involves activation of the carboxylic acid of PLGA by DCC and NHS, and the formed O-acyl derivative of carboxylic acid is more prone to reaction with a nucleophile (Valeur and Bradley, 2009), the i.e. amine group of gemcitabine, which leads to amide bond formation between gemcitabine and PLGA Characterization by analytical methods Various analytical techniques were utilized for characterization of polymeric conjugate. The FT-IR spectra (Fig.17) of gemcitabine showed characteristics bending vibrations of amines at 1702 cm -1 and 1658 cm -1 and stretching vibration of amine at 3261 cm 1 which are in accordance with previous reports (Cavallaro et al., 2006; Tao et al., 2012). In the FT-IR spectra of pure PLGA, the peak at 1753 cm -1 is exhibited due to the absorbance of the carbonyl group in PLGA matrix, and peaks at 2998 cm -1, 2953 cm -1, and 2853 cm -1 correspond to (C-H) bending vibrations (Deniz, 1999; Stevanovic et al., 2008). On the other hand, the FT-IR the spectrum of PLGA gemcitabine conjugate showed the characteristic peak of the carbonyl group of PLGA at 1750 cm -1 and C-H bands at 2954 cm -1, 2924 cm -1, and 2854 cm -1. The peaks at 1665 cm -1 and 1536 cm -1 confirm the formation of an amide bond between PLGA and gemcitabine. PLGA gemcitabine conjugate showed a peak at 3417 cm -1 attributed to stretching (-N-H) vibration band of secondary amide (Cavallaro et al., 2006; Chitkara et al., 2013; Tao et al., 2012). The polymeric conjugate of gemcitabine was also characterized by 1 H NMR (Fig.18). The recorded 1 H NMR spectra of pure PLGA showed the typical signal approximately at 1.48 ppm, attributed to the methyl groups of the D- and L-lactic acid monomers, and the multiplets around 5.23 and 4.92 ppm, due to the CH groups of lactic acid and CH2 groups of glycolic acid, respectively. The findings are in accordance to previous investigations (Cenni et al., 2012; (Pignatello et al., 2009). The 1 H NMR spectra of gemcitabine give the signal for the protons of

2 5 and 3 OH groups at 6.05 ppm and 6.16 ppm, respectively (Cavallaro et al., 2006; Pasut et al., 2008a). Figure 1: Fourier transforms infrared spectra of PLGA, gemcitabine, and PLGA- gemcitabine conjugate (PLGA-GEM) in the region 4000 to 500 cm -1. The signal of 4 -NH2 group of gemcitabine was registered at 8.07 ppm, which is in accordance with a previous report (Vandana and Sahoo, 2010). On the other hand, 1 H NMR spectra of PLGA gemcitabine conjugate showed a signal of methyl groups of PLGA at 1.48 ppm and CH of lactic acid and CH2 of glycolic acid at 5.18 ppm and 4.8 ppm, respectively. Further, 1 H NMR spectra also showed the chemical shifts at 6.17 ppm and 6.34 ppm corresponding to 5 - and 3 -OH groups. The appearance of a signal at 8.29 ppm confirms the formation of amide

3 bond which is shifted from 8.07 of the amine group of gemcitabine (Bondioli et al., 2010; Vandana and Sahoo, 2010). Figure 2: 1 H- NMR (500 MHz) spectra of PLGA, gemcitabine and PLGA-gemcitabine conjugate (PLGA-GEM) in deuterated DMSO In vitro stability and release profile of gemcitabine in plasma The rate of gemcitabine release from polymeric conjugate and its stability in plasma are important considerations as the drug is metabolized to its inactive form when administered intravenously. The rate of release of the gemcitabine in plasma from the PLGA-gemcitabine conjugate is shown in Fig.19. Initially, during the 1 h incubation period, approximately 16% of the gemcitabine was available in plasma. Further, after 72 h the release from polymeric conjugate increases and about 40% of gemcitabine was available in plasma, whereas in the case of native gemcitabine only 7% of the drug was detected at this time point. The availability of gemcitabine in the plasma suggested that after incubation in plasma the drug in the polymeric

4 % Drug Present in Plasma conjugate was more stable as compared to native gemcitabine. It was observed that more than 50% of the native gemcitabine was degraded in just 1 h and the negligible amount was detected after 72 h. The results showed that the stability of gemcitabine in the plasma was improved following conjugated with PLGA GEM PLGA-Gemcitabine Time (hrs) Figure 3: Time-dependent availability of gemcitabine in plasma when released from PLGAgemcitabine (2.4 mg/ml) and stability of native gemcitabine (0.2 mg/ml) in plasma In vitro cytotoxicity The gemcitabine and PLGA-gemcitabine conjugate were tested for their in vitro cytotoxicity in a panel of cell lines including MIAPaCa-2, MCF-7, and HCT-116 at different concentrations for 72 h (Fig. 20). In HCT-116 cell line, PLGA-gemcitabine conjugate showed similar activity to native gemcitabine, whereas in MCF-7 and MIAPaCa-2 cell lines, PLGA-gemcitabine conjugate showed an enhanced efficacy than native gemcitabine. An IC50 of 2.4 ng/ml and 2.8 ng/ml were observed in HCT-116 cell line for native gemcitabine and PLGA-gemcitabine conjugate, respectively. In MCF-7 cell line, IC50 in the order of 132 ng/ml and 15 ng/ml were observed for native gemcitabine and PLGA gemcitabine conjugate, respectively. Similarly, MIAPaCa-2 cell line displayed higher cytotoxicity for PLGA-gemcitabine conjugate with IC50 value in the order of 7.5 ng/ml as compared to 24 ng/ml for native gemcitabine. The cytotoxic activity of the conjugate could be related to the improved lipophilicity of the gemcitabine the

5 % Cell Inhibition % Cell Inhibition % Cell Inhibition following conjugation with PLGA, which could enhance the intracellular uptake and in turn, attributed to improved anti-proliferative effect GEM HCT GEM MCF-7 * 80 PLGA-GEM 80 PLGA-GEM * * Conc. μg/ml Conc. μg/ml MIAPaCa-2 GEM PLGA-GEM * * * * Conc. μg/ml Figure 4 : Percent cell inhibition on different concentrations of gemcitabine and PLGAgemcitabine conjugate (PLGA-GEM) against different cell lines. Cells were incubated with gemcitabine and PLGA-GEM conjugate for 72 h. The results represent mean ± S.D of three separate experiments. *P<0.05 vs GEM Nucleoside transporter inhibition The gemcitabine, being a hydrophilic compound, does not readily cross the cell membrane by diffusion (Mackey et al., 1998a). This necessitates the presence of specialized transport protein such as human equilibrative nucleoside transporters (hent1 or hent2) for efficient transport of gemcitabine (Damaraju et al., 2003). It has been demonstrated that deficiency in hent1 confers resistance to gemcitabine toxicity in vitro (Possinger, 1995). Therefore in order to investigate whether the PLGA-gemcitabine conjugate also relies on one or more of these transporters, the nucleoside transporter inhibition study was performed. Dipyridamole (a nucleoside transporter inhibitor) was used 30 min before the addition of

6 varying concentrations of native gemcitabine and PLGA-gemcitabine conjugate. The IC50 values were obtained for the human pancreatic cancer cell line, MIAPaCa-2. In MIAPaCa-2 IC50 of 464 ng/ml was obtained for native gemcitabine in the presence of dipyridamole which is approximately 19 folds greater than the value when cells were not pre-incubated with the nucleoside inhibitor. However, for PLGA-gemcitabine conjugate an IC50 of 7.0 ng/ml was observed which is similar to the values when cells were not pre-incubated with the nucleoside inhibitor (Table 5). Alternatively, it can be stated that in MIAPaCa-2 cell line, dipyridamole markedly decreased sensitivity to native gemcitabine, while sensitivity to PLGA-gemcitabine conjugate was not altered. The results suggest that gemcitabine activity was many folds lower when pre-incubated with dipyridamole, however, the similar cytotoxic effect was observed in the case of PLGA-gemcitabine conjugate, suggesting that they do not enter the cells via the nucleoside transporters. Table 1 : The effect of absence and presence of nucleoside transporter inhibitor on the antiproliferative activity of gemcitabine and PLGA-gemcitabine after incubation of 72hrs. Compounds MiaPaCa-2 Relative Gem Resistance Gemcitabine (IC50) 0.024±0.005 Gemcitabine + Dipyridamole (IC50)* ± PLGA-Gemcitabine (IC50) ± PLGA-Gemcitabine +Dipyridamole (IC50) 0.007± *Statistical significant (p<0.005 Vs Gemcitabine) Cell cycle analysis In order to investigate the cell death caused by native gemcitabine and PLGA-gemcitabine conjugate in MIAPaCa-2 cells, the cell cycle analysis using propidium iodide staining was performed. The DNA-specific fluorochrome propidium iodide discriminates apoptotic cells (sub-g1 cell population) wherein apoptosis-associated endonucleases degrade the DNA. By measuring the DNA content using flow cytometry, the sub-g1 cell population of 72 h gemcitabine and PLGA-gemcitabine conjugate treated MIAPaCa-2 cells were analyzed. The percentage of apoptotic cells was greater in PLGA gemcitabine conjugate than native gemcitabine. The sub G1 (G0) apoptotic population was observed to be 46.6% and 60.6% for native gemcitabine and PLGA-gemcitabine conjugate, respectively, as compared to 3.55% in control cells (Fig. 21).

7 % Cell Population ve control Gemcitabine PLGA-GEM * * sub G1 G1 S G2/M Different Phase of Cell Cycle * Figure 5: Effect of gemcitabine and PLGA-gemcitabine conjugate (PLGA-GEM) on cell cycle distribution of human pancreatic cancer cell (MIAPaCa-2). Flow cytometric analysis of MIAPaCa-2 cells was performed after propidium iodide staining. Cells were incubated for 72 h in the presence of gemcitabine (0.25µg/ml) and PLGA-gemcitabine conjugate (0.25µg/ml). Figure shows the representative mean±sd of percent population of cells in each phase of cell cycle of three individual experiments.*p<0.05 vs GEM Mitochondrial membrane potential At the induction of apoptotic condition, the inner mitochondrial transmembrane potential is disrupted, eventually leading to outer mitochondrial membrane rupture and release of proapoptotic proteins into the cytoplasm. Rh-123, a cationic fluorophore, is actively accumulated by cells and is useful in monitoring the membrane potential of mitochondria. The energization of mitochondria induces quenching of Rh-123 fluorescence and is directly proportional to mitochondrial membrane potential. It has already been reported that gemcitabine-induced apoptosis through MMP loss (Bortner and Cidlowski, 1999). Therefore the effect of gemcitabine and conjugated gemcitabine was observed on MIAPaCa-2 cells at a concentration of 0.25 µg/ml following incubation for 72 h. The untreated cells showed 8.4% loss of MMP after 72 h incubation. The gemcitabine showed 25.9% loss of MMP whereas 42.1% MMP loss was observed for PLGA-gemcitabine conjugate (Fig. 22). These results indicated that

8 PLGA-gemcitabine conjugate induced greater cell death implicated through greater loss of MMP in comparison to native gemcitabine. Figure 6: Effect of gemcitabine and PLGA-gemcitabine conjugate on mitochondrial membrane potential loss in human pancreatic cancer cell line (MIAPaCa-2). Cells were incubated at equivalent gemcitabine concentration of 0.25µg/ml in 6 well plates for 72 h treatment. Figures show the representative staining profile of one of two similar experiments Western blot analysis To maintain homeostasis in normal cells apoptosis plays a major role. Apoptosis is characterized by various morphological changes including chromatin condensation, cell shrinkage, membrane blebbing and changes at the molecular level including internucleosomal DNA fragmentation and cleavage of poly (ADP-ribose) polymerase-1 (PARP-1). PARP-1 plays an important role in repairing damaged DNA. It is activated, and its level increases when DNA damage is induced in cells. It contains an active site and a DNA binding domain that are separated on cleavage into 89 kda and 24 kda fragments by caspase-3 following induction of apoptosis (Kaufmann et al., 1993). The western blot of PARP cleavage in MIAPaCa-2 cancer cell line was performed following 72 h of native gemcitabine and PLGA-gemcitabine conjugate treatment. The PARP cleavage was observed in gemcitabine and PLGA-gemcitabine conjugate, with latter showing greater cleavage in comparison to the native gemcitabine. The western blot analysis of P-H2AX further corroborated these results. The histone variant H2AX is phosphorylated at Ser 139 (g- H2AX) in response to DNA damage during apoptosis. P-H2AX senses the DNA double strand breaks and serves as a docking site for DNA damage checkpoint protein 1 (facilitates recruitment of various proteins for DNA repair) at the damage foci, thus is important for initiating early DNA damage response (Sharma et al., 2012). The gemcitabine inhibits DNA

9 synthesis, causing DNA damage thus elevating the levels of P-H2AX. The western blot analysis of P-H2AX in MIAPaCa-2 following 72 h of gemcitabine and PLGA-gemcitabine treatment revealed greater expression of P-H2AX in response to PLGA-gemcitabine conjugate in comparison to native gemcitabine. The results suggested enhanced efficacy and biological action of PLGA-gemcitabine conjugate than the native gemcitabine (Fig. 23). Figure 7 (A) Protein expression of PARP and P-H2AX detected by western blot of the MIAPaca- 2 cells treated with gemcitabine and PLGA-gemcitabine conjugate (PLGA-GEM). Cells were treated with equivalent gemcitabine concentration of 0.25µg/ml for 72h. An equal amount of the proteins were fractionated on SDS-polyacrylamide gels and transferred to nitrocellulose membrane, followed by immunoblotting with anti-parp and anti-p-h2ax antibodies. β-actin is shown as a loading control. (B) Densitometric analysis of cleaved PARP and the P-H2AX expression on untreated cells, gemcitabine-treated cells and PLGA-GEM treated cells. *P<0.05 vs GEM. 4.2 Synthesis of TPGS-gemcitabine micelles The TPGS-Gem micelle was prepared after synthesis of TPGS-Gem prodrug. The gemcitabine was conjugated to TPGS through a succinate linker as a two-step process: (I) modification of end group of TPGS to a carboxylic group using succinic anhydride and (II) coupling of activated ester form of succinic acid-modified TPGS to the amino group of gemcitabine using classical

10 carbodiimide chemistry. All the reactions performed to synthesize TPGS-Gem prodrug were characterized by different analytical techniques including FT-IR, 1 H-NMR, and MALDI-TOF. 4.3 Characterization of TPGS-Gem prodrug Fourier transform infrared spectra The FT-IR spectra of native TPGS (Fig. 24) showed a characteristic band at 1736 cm -1 which is attributed to the carbonyl group present in the TPGS. In the FT-IR spectra of TPGS-SA, the strong absorption of carbonyl band at 1732 cm -1 was observed, which confirmed the formation of TPGS-SA, these findings are in accordance with previous reports (Bao et al., 2014). Moreover, in the FT-IR spectra of gemcitabine, the band found at 1674 cm -1 and 1662 cm -1 attributed as a characteristic amine bending vibrations. In contrast, the FT-IR spectra of TPGS-Gem prodrug showed the characteristic carbonyl band at 1734 cm -1 in addition, bands at 1654 cm -1 and 1560 cm -1 represents amide I (C=O, stretching) and amide II (N-H bending), respectively, confirmed the formation of amide bond between terminal carboxyl group of TPGS and primary amino group of gemcitabine (Khare et al., 2014).

11 Figure 8 : Stacked Fourier transform-infrared (FT-IR) spectra of TPGS, gemcitabine, and TPGS-Gem prodrug scanned in the region of 3600 cm -1 to 800 cm Nuclear magnetic resonance The structure of TPGS-Gem prodrug was also characterized by 1 H NMR spectra (Fig. 25). In the 1 H NMR spectrum of TPGS, the signals of ethylene protons of PEG chain, present in TPGS were observed at δ The signals registered in the aliphatic region (δ1-3) of spectra attributed to the various protons of Vitamin E-tail. 1 H NMR spectra of TPGS-SA showed similar signals as observed in the spectra of TPGS except succinyl methylene (-CH2) protons at δ , confirmed the reaction between TPGS and succinic anhydride (Guo et al., 2013). In the 1 H NMR spectrum of gemcitabine, peaks at δ 8.2 correspond to the protons of a 4-amino group of gemcitabine. Other characteristic signals of 5 and 3 OH of gemcitabine was registered at δ 6.08 and 6.28 respectively (Tao et al., 2012). The 1 H NMR spectra of TPGS-Gem prodrug showed the characteristic ethylene protons of PEG chain at δ 3.5 with various protons of vitamin E-tail. However, the peak of the amino group of gemcitabine was shifted from δ 8.2 to δ 11.2 suggested the formation of an amide bond between gemcitabine and TPGS (Maurizio et al., 2015; Tao et al., 2012). Figure 9: Stacked 1 H-NMR (400 MHz) spectra of TPGS, TPGS-SA, gemcitabine, and TPGS- Gem micelles registered using trimethylsilane as an internal standard.

12 4.3.3 MALDI-TOF mass spectroscopy The MALDI-TOF spectra of TPGS, TPGS-SA and TPGS-Gem was investigated to assign the formula of various oligomeric compositions with different adduct and charge states (Fig. 26). The molecular formula of TPGS forming potassium adduct was validated as C33H54O5- (CH2CH2O)n + K. In the MALDI spectrum of TPGS-SA, a marked 100 Da increment in the mass of various oligomers was observed, which is equal to the mass of succinic anhydride, suggesting the reaction between TPGS and succinic anhydride. An expected empirical formula for various oligomers of TPGS-SA forming potassium adduct could be [C33H53O5- (CH2CH2O)n]-CO-C2H4-COOH+K. Moreover, the MALDI spectra of TPGS-Gem prodrug showed an increment of 245Da in oligomeric clusters as compared to the oligomers of TPGS- SA, which is similar to the increment of mass if gemcitabine conjugated to TPGS-SA via an amide bond. However, there was no adduct (including K, Na) was seen in the oligomeric clusters of TPGS-Gem spectra. Furthermore, in the spectra of TPGS and TPGS-SA, the oligomers present in different charge state. For instance, the reaction between TPGS oligomer with n-value 20, [C33H54O5- (CH2CH2O)20, 1411 Da; or m/z 1450 in the potassium salt form] occurs with succinic acid would lead to the species at m/z 1511 (or m/z 1550 in the form of potassium salt) and further reaction with gemcitabine would lead to the species at 1757 (with no adduct formation). By comparing the spectra of TPGS, TPGS-SA and TPGS-Gem an increment of the abundance of the ion at 1550 in TPGS-SA and 1757 in the spectra of TPGS-Gem is clearly visible, confirmed the transformation of the terminal hydroxyl group to the carboxyl group at TPGS terminal end after I step of reaction and formation of an amide bond between TPGS-SA and gemcitabine after II step. The similar behavior was noticed for all other oligomeric species. The concise table of proposed formulas of oligomeric compositions of TPGS-Gem prodrug and intermediates is given in Table-6. The conjugation of gemcitabine to TPGS is an important consideration since gemcitabine as a hydrophilic drug have very low encapsulation efficiency in polymeric NPs. TPGS-Gem micelles could be used as a new strategy to deliver hydrophilic drug if the loading efficiency is high. The amount of drug conjugated to TPGS was calculated to be 36.3 ± 2.5 µg/mg of TPGS-Gem prodrug.

13 Figure 10: Full mass overview spectra s of TPGS (A), TPGS-SA (B) and TPGS-Gem prodrug (C) by MALDI-TOF.

14 Table 2: Comparison of theoretical molecular weight with the experimental molecular weight of TPGS, TPGS-SA, and TPGS-GEM obtained in MALDI Spectra's. TPGS TPGS-SA TPGS-Gemcitabine PEG units Theoretical Experimental Theoretical Experimental Theoretical Experimental n= n= n= n= n= n= n= n= n= n= n= n= n= Micelle generation and characterization The TPGS-Gem micelles were generated by simple solvent evaporation method as described in section The amphiphilic TPGS-Gem prodrug generates micelles by self-assembly in aqueous solution. After evaporation of the solvent, the generated micelles were characterized using various physicochemical parameters Particle size, zeta-potential and morphological analysis The average particle diameter of TPGS-Gem prodrug was investigated upon nano-aggregation into micelles at different temperatures (25, 30, 35, 40 0 C). No significant alteration was observed in the hydrodynamic diameter at selected temperatures, suggested that temperature does not influence the average particle size of micelles. An average particle size at 25 0 C was measured as ± 0.7 nm (Fig. 27A) with a polydispersity index, ± Further at higher temperature the change in the size of TPGS-Gem micelles has been observed to be negligible and the average hydrodynamic was registered as 15.67±0.16, 15.38±0.34, 15.94±0.59 and PDI 0.19±0.02, 0.115±0.037, 0.153±0.032 respectively, at temperature 30 0 C, 35 0 C, and 40 0 C. The zeta potential of the micelles was evaluated as -9.3 ± 0.61 mv (Fig. 27B). Particle size and zeta potential are the key factors for achieving the therapeutic efficacy of nanomedicines and micelles typically between 10 nm-100 nm are known to be favorable for

15 drug delivery in cancer therapy. Particles smaller than 10 nm in size will be rapidly abolished through renal clearance (threshold < 6 nm) and greater than 100 nm, have a probability to uptake by the reticuloendothelial system (RES) (Blanco et al., 2009). Moreover, either neutral or anionic surface charge is considered beneficial for nanocarriers for successful evasion of renal elimination could result in long circulatory action in vivo (Acharya and Sahoo, 2011). The transmission electron microscopy (TEM) was used to determine the morphology of TPGS-Gem micelles; from which it can be seen that micelles have generally spherical shape (Fig. 27C), however, a slightly bigger than the particle size was observed with DLS. The difference in the size obtained by two techniques could be due to the melting property of TPGS (melting point 38 0 C). The micelles experience a certain extent of melting and expansion in size due to a high energy electron beam in TEM which makes them appear bigger in the images captured by TEM (Mi et al., 2012). Figure 11: Representative particle size distribution (A) and zeta potential (B) of TPGS-Gem micelles measured by dynamic light scattering measurement at 90 0 angle at 25 0 C using disposable polystyrene cells and folded capillary cells respectively. (C) Transmission electron micrograph of TPGS-Gem micelles revealing size of micelles captured using a formvar-coated copper grid (D). Critical micelle concentration measurement of TPGS-Gem micelles using methyl orange as a hydrophobic probe, an inflexion point in the graph represents the CMC value.

16 4.4.2 Critical micelle concentration The CMC value of TPGS-Gem prodrug and native TPGS was investigated using methyl orange as a hydrophobic probe in aqueous solution by monitoring the hypsochromic shift in methyl orange UV spectra (Fig. 27D). Methyl orange has λmax at 464 nm in the presence of UV light. When a surfactant was diluted in methyl orange solution, it favors the hydrophobic core formed from the micelles of the amphiphilic polymers and a hypsochromic shift was experienced at critical micelle concentration value on UV spectra of amphiphilic polymer or surfactant (Lin et al., 2012). The effect of concentration of TPGS-Gem prodrug on the peak absorption of methyl orange at λmax 464 nm is given in the Fig. 27D. The native TPGS exhibited the CMC value at 0.2 mg/ml (0.02%) (Constantinides et al., 2006; Kulhari et al., 2015) and it was observed that the CMC value of TPGS-Gem prodrug was 0.15 mg/ml (0.015%). A remarkable decrease in the CMC value of TPGS-Gem was observed, attributed to the modification with a hydrophilic moiety. The lower CMC value of micelles indicated high stability, which prevents its dissociation into unimers upon dilution with large blood volume In-vitro drug release The rate of gemcitabine release from TPGS-Gem micelles was evaluated in PBS buffer (ph 7.4) at various temperatures (30, 37 and 40 0 C). As illustrated in Fig. 28 the release of gemcitabine from prodrug micelles showed significantly slower release at 30 0 C than that at 37 and 40 0 C. However, a lower increment of drug release at temperature 40 0 C was observed than that of drug release at 37 0 C. Under all temperatures, the release of gemcitabine began as an initial slow release (2.4% at 30 0 C, 8.5% at 37 0 C and 10% at 40 0 C in 1 h) followed by a continued sustained release and exhibited a similar release pattern at all the temperatures initially. The release of gemcitabine after 48 h was 38 % at 30 0 C, 59 % at 37 0 C and 64 % at 40 0 C and a sustained released was observed subsequently. The accumulative release at one week was measured as 58% at 30 0 C, 89% at 37 0 C 59% and 95% at 0 C. The drug release study demonstrated that TPGS-Gem micelles were able to sustain gemcitabine release at selected temperatures.

17 Figure 12: Release profile of gemcitabine from TPGS-Gem micelles after incubation in PBS (ph 7.4) for one week, at various temperatures i.e. 30, 37 and 40 0 C. The concentration of drug released at specific time point was analyzed validated HPLC method. (*P < 0.05, 30 0 C vs 37 0 C /40 0 C, after Bonferroni correction, n=3) Stability studies with cytidine deaminase After intravenous administration, gemcitabine undergoes extensive metabolism to its inactive form, either intracellular or extracellular, due to the activity of CDA (Abbruzzese et al., 1991). The stability of gemcitabine in the micellar formulation was investigated after incubation with crude CDA in comparison with parent gemcitabine. The concentration of drug was quantified at various time intervals using LC-MS/MS. As a function of incubation time with cytidine deaminase, the concentration of gemcitabine is shown in Fig. 29. The disappearance in the native gemcitabine was observed time-dependently and only around 11% of gemcitabine was detected after 30 min of incubation. Whereas, highly significant resistant was observed in the gemcitabine micellar formulation with more than 90% of gemcitabine can be detected after 30 min of incubation with crude CDA. This modest degradation of gemcitabine in TPGS-Gem micelles demonstrated the stability of formulation against CDA. These results are in accordance with previous reports, suggested that substitution of a 4-(N) amino group of gemcitabine prevents enzymatic degradation (Tao et al., 2012), (Song et al., 2005).

18 Figure 13: Enzymatic stability of TPGS-Gem micelles in comparison to native gemcitabine. Micellar gemcitabine or free gemcitabine (0.4 mm) was incubated in a buffer (ph 7.5) containing cytidine deaminase (10µl, specific activity 3.5unit/mg) at 37 C. (*P < 0.05, n=3) In vitro cytotoxicity The in vitro antiproliferative activity was performed by standard MTT assay after incubation of TPGS-Gem micelles, gemcitabine and placebo TPGS micelles with BxPC-3 cells as a model pancreatic cancer cell line at various incubation points i.e. 24 h, 48 h, and 72 h (Fig. 30 A, B, C respectively). Results suggested that the various test samples exhibited dose and time dependent cytotoxicity against BxPC-3 cells. It is noteworthy that placebo TPGS micelles also showed significant antiproliferative activity. The TPGS-Gem micelles and placebo TPGS micelles showed highly significant growth inhibition of BxPC-3 cells after 24 h as compared to gemcitabine. The IC50 value of native gemcitabine was evaluated as 165 ± 8.65 µm at 24 h of incubation time while the IC50 value of TPGS-Gem micelles was 6.13 ± 0.35 µm, and the IC50 value of the equivalent amount of placebo TPGS micelles was found to be 9.09 ± 0.7 µm. Further, after 48 h of incubation with samples, the same trend was noticed and TPGS-Gem micelles showed a significant higher cytotoxicity as compared to gemcitabine, however, the IC50 value of gemcitabine decreased massively to 2.4 ± 0.23 µm, and the IC50 value of TPGS-Gem micelles and placebo TPGS micelles was demonstrated as 1.6 ± 0.26 µm, 3.1 ± 0.28 µm, respectively. A further increase in the incubation time to 72 h, result in further decrease in the drug concentration required to 50% of growth inhibition. The IC50 values were decreased to

19 0.27 ± 0.1 µm, 0.6 ± 0.22 µm, and 1.76 ± 0.22 µm for gemcitabine, TPGS-Gem micelles, and placebo TPGS micelles respectively. However, there was a comparable difference in the IC50 value of gemcitabine and TPGS-Gem micelles after 72 h of incubation (Fig. 30D). The enhanced cytotoxic efficacy of gemcitabine could be due to the intrinsic anticancer efficacy of TPGS and/or stability of gemcitabine in micellar formulation against cytidine deaminase, which can convert the monophosphorylated form of gemcitabine to an inactive metabolite. Figure 14: Percent of cell viability on different concentrations of gemcitabine, TPGS-Gem micelles and placebo TPGS micelles (i.e. TPGS with no drug conjugated, at equivalent weight as in TPGS-Gem micelles) against BxPC-3 cells at incubation time 24h (A), 48h (B) and 72h (C). The IC50 value obtained cells after incubation of samples at different time points (D). The results represent mean SD of three experiments. *P < 0.05 vs. Gemcitabine/ Placebo TPGS micelles Nucleoside transporter inhibition Due to the highly hydrophilic character of gemcitabine, it relies on the nucleoside transporter to cross the cellular lipid bilayer and display its antiproliferative activity. A resistance mechanism of gemcitabine includes deficiency of nucleoside transporters, causes a reduction in the transport

20 activity of drug into the cell (Bergman et al., 2002). In order to examine, whether TPGS-Gem micelles also dependent on nucleoside transporters like gemcitabine, a nucleoside transporter inhibition assay was performed. During MTT assay, the cells were treated with dipyridamole, to block the nucleoside transporter, prior to addition of various concentration of TPGS-Gem micelles and gemcitabine. The IC50 values were determined in BxPC-3, pancreatic cancer cells after 48h of incubation (Table 6). The IC50 value of native gemcitabine obtained after treatment with dipyridamole was 11 µm, which is 5-fold higher than the IC50 value obtained with the cell not treated with dipyridamole. However, when TPGS-Gem micelles were treated with cells preincubated with dipyridamole, there was no significant change was observed in the IC50 value. These results reflect that TPGS-Gem micelles were not dependent on nucleoside transporter to cross the cellular lipid bilayer. Table 3: Effect of nucleoside transporter inhibitor on the anti-proliferative activity of gemcitabine and TPGS-gem micelles against BxPC-3 cell lines following 48h of incubation. Sample IC50 (µmol) BxPC-3 Cells Relative gemcitabine resistance Gemcitabine Gemcitabine + dipyridamole TPGS-Gem micelles TPGS-Gem micelles + dipyridamole Cell Uptake The qualitative uptake of coumrin-6 loaded TPGS-Gem micelles in BxPC-3 cells was determined in the presence and absence of dipyridamole by measurement of fluorescence. Coumarin-6 was used as a fluorescent marker to identify the uptake of micelles in BxPC-3 cells. Fig. 31 shows the uptake of TPGS-Gem micelles after 4 h of incubation with BxPC-3 cells, the green fluorescence was due to the coumarin-6 loaded micelles internalized by the cells. In order to confirm that fluorescence was present inside the cells, the nucleus was counterstained with a DAPI, a blue fluorescent dye. The green fluorescence adjacent to the nucleus confirming the micelles were within the cells instead of attached to the cell surface. Furthermore, nearly all cells either in presence (Fig. 31A) and absence (Fig. 31D) of nucleoside transporter inhibitor were stained with green fluorescence suggesting that there was no significant difference in the

21 uptake of micelles, suggesting that in the micellar formulation gemcitabine does not dependent on nucleoside transporters for internalization in the cells. These results can be correlated with the insignificant change in the IC50 values obtained after inhibition of nucleoside transporters by dipyridamole. Figure 15 : Demonstration of cellular uptake of TPGS-Gem micelles using fluorescence microscopy in BxPC-3 cell lines in the presence (A-C) and absence (D-F) of nucleoside transporter, dipyridamole. Micelles uptaken by cells showing green fluorescence and nuclei stained with DAPI showing blue fluorescence. Images were captured using EVOS Floid cell imaging station AFM topography To study the morphological changes in BxPC-3 cells after treatment with TPGS-Gem micelles and native gemcitabine, the cells were observed under atomic force microscope (AFM). The control cells were treated with media only. It was observed that the control cell had a smooth well-defined morphology and showed an epithelial cell shape in general (Fig. 32A). The AFM image of cell treated with native gemcitabine showed a smooth surface, however, marked specific degenerative alteration in the cell shape can be easily observed in the image (Fig. 32B). This could be correlated with the higher IC50 value of the gemcitabine after 24 h of incubation. In the AFM image of TPGS-Gem micelles treated a cell, numerous pits on the surface is clearly visible and the morphology of cell was also transformed (Fig. 32C). The small pits on the cells attributed the morphological alteration in response to TPGS-Gem micelles. The ability to

22 visualize cellular state is advantageous in order to accurately demonstrate the effect of these formulations on the morphology and physiology. Although not qualitative these provide a good tool to compliment the cytotoxicity studies and provide insight into the likely fate of the cells. In this study, the presence of pitting indicated that cellular morphology has been altered and perhaps cell membrane integrity has been compromised. This indicates that the cells are not residing in their normal state. Hence, the observations of AFM topography suggested the enhanced efficacy of gemcitabine after formation of TPGS conjugated micelles (Hoskins et al., 2012). Figure 16 : Effect of gemcitabine and TPGS-Gem micelles on the cellular morphology in human pancreatic cancer cell line (BxPC-3) by AFM topography. The cells were treated with a 10µmol concentration of test sample with respect to drug concentration in 6 well plates for 24h and fixed by 2.5% of glutaraldehyde before images were captured under AFM. (A) non-treated control cell, (B) cell treated with gemcitabine and (C) cell treated with TPGS-Gem micelles (arrow showing the pits on the surface of the cell) 4.5 Synthesis and characterization of mpeg-plga di-block In this study, pegylated NPs of gemcitabine were developed, where PEG served as the hydrophilic shell and the hydrophobic core is composed of PLGA. The terminal carboxyl group of PLGA was conjugated with methoxy-peg-amine using DCC/NHS based coupling. The formation of mpeg-plga copolymer was validated by FT-IR and NMR.

23 4.5.1 FT-IR In the FT-IR spectra of PLGA, the peak at1753 cm -1 was exhibited due to the absorbance of the carbonyl group in PLGA matrix and peaks at 2988 cm -1, 2953 cm -1 and 2853 cm -1 corresponds to (C-H) bending vibrations (Fig. 33). IR spectra of PEG exhibited characteristic peaks at 1098 cm -1 (CH2-O-CH2), 3435 cm -1 (N-H), 2924 cm -1 (C-H stretching). The conjugation between PLGA and PEG was confirmed by the presence of amide band I at 1673 cm -1 and amide II at 1540 cm -1 suggested to C=O stretching and N-H bending of amide respectively. All the characteristic peaks of PLGA and mpeg amine including carbonyl group at 1759 cm -1, 2927 cm -1 (C-H stretching) and 1095 cm -1 (CH2-O-CH2) was also observed in mpeg-plga infrared spectra. Figure 17: Fourier transform infrared spectra of PLGA, mpeg and mpeg-plga scanned in the region of 4000 to 500 cm -1 using chloroform as a solvent H NMR The recorded 1 H NMR spectra of PLGA exhibited characteristic signals at 1.48 ppm, due to the methyl groups of the D-and L-lactic acid monomers, and the multiplets around 5.23 and 4.92

24 ppm, attributed to the CH groups of lactic acid and CH2 groups of glycolic acid, respectively (Fig. 34). Whereas mpeg-plga conjugate showed characteristics peak of PLGA and singlet at 3.7 ppm (CH2-CH2-O)n as the characteristic peak of PEG confirming the formation of mpeg- PLGA conjugate. The concentration of PEG in mpeg-plga copolymer was determined by a BaCl2/I2 calorimetric method. The content of PEG was 9.15% ± 0.6% (26.9µmol/gm of polymer) of the total weight of the synthesized copolymer. This level of PEG content is suitable in view of the fact that PLA-PEG copolymer with 10% of PEG exhibited minimum uptake by macrophages (Sheng et al., 2009). Figure 18: Proton NMR (400 MHz) spectra of PLGA, mpeg-plga in CDCl3, recorded using trimethyl silane as an internal standard. 4.6 Characterization of mpeg-plga NPs Size, zeta potential, morphology, and drug loading The size of the NPs should be adequately small to avoid the detection by reticuloendothelial system in order to attain longevity in the systemic circulation. The average particle size,

25 polydispersity index and zeta potential of various drug/copolymer ratios of pegylated and nonpegylated NPs are given in Table 8. The highest loading efficiency was obtained in the drug/copolymer ratio 1:5 and 1:7. However there is no significant difference in the loading efficiency of these two ratios. Therefore, we have considered drug/copolymer ratio of 1:7 as an optimized formulation and all further investigations was performed using NPs prepared by same drug/copolymer ratio i.e. 1:7. The pegylated NPs exhibited drug loading as 7.07 µg which is significantly higher compared to the non-pegylated counterpart with same drug/copolymer ratio 4.53 µg (Table 8). The higher loading in pegylated NPs could be credited to the hydrophilic characters of PEG that creates the core-shell structure of the NPs, which help to prevent diffusion of the drug during the preparation process. The size of the NPs was found to be suitable for drug delivery to the solid tumor Fig. 35 (A-D). The morphology of non-pegylated and pegylated NPs was investigated by TEM (Fig. 35 E-F). It was observed that NPs were distributed as individual particles with a distinct spherical core-shell arrangement dispersed uniformly about 200 nm in size. The TEM image clearly showed the non-pegylated NPs with a dark core attributed to PLGA matrix, while pegylated NPs exhibited a dark core surrounded by a lighter shell of PEG layer, which is in agreement with a previous finding (Wang et al., 2011). Table 4: Characteristics of pegylated and non-pegylated NPs Nanoparticles Ratio Size (nm) Polydispersity Index Zeta potential (mv) Loading (µg/mg) mpeg-plga NPs 1:5 272 ± ± ± ± 0.11 mpeg-plga NPs 1:7 267 ± ± ± ± 0.35 mpeg-plga NPs 1: ± ± ± ± 0.81 mpeg-plga NPs 1: ± ± ± ± 1.02 mpeg-plga NPs 1: ± ± ± ± 0.4 PLGA NPs 1:7 268 ± ± ± ± 0.17

26 Figure 19: Representative particle size of non-pegylated (A) and pegylated NPs (B) recorded by dynamic light scattering measurement at 90 0 angle using disposable polystyrene cells and zeta potential of non-pegylated (C) and pegylated NPs (D) recorded by folded capillary cells. Transmission electron microscopic images revealing the size and shape of non-pegylated (E) and pegylated (F) gemcitabine encapsulated NPs.

27 4.6.2 In-vitro drug release profile The in-vitro release of gemcitabine from NPs was evaluated in PBS buffer (ph 7.4) at 37 o C. The non-pegylated NPs and pegylated NPs exhibited a similar release pattern of gemcitabine, which began as initial burst release followed a sustained release pattern (Fig. 36). The release of gemcitabine was rapid in the initial 12 h in order of 42% and 49% for non-pegylated and pegylated NPs respectively and thereafter a sustained released was observed. The accumulative release from non-pegylated and pegylated NPs after 48h was 77±2.6% and 68±2.5% respectively. Figure 20 : Release profile of gemcitabine from pegylated and non-pegylated NPs after incubation for up to 48 h in PBS (ph 7.4) at 37 0 C. The concentration of drug released at specific time point was analyzed by validated HPLC method Potential for hemolysis The assessment of the compatibility with blood component is an important aspect for intravenously administered formulations. Therefore, gemcitabine loaded pegylated and nonpegylated NPs were evaluated in vitro for hemolytic activity with red blood cells. Hemolysis was determined by quantifying the amount of the released hemoglobin following incubation with the sample. The concentration of the pegylated and non-pegylated NPs was equivalent to the 2mg/kg of gemcitabine, the dose which was used in the in-vivo experiment. The hemolytic potential of the copolymer was compared with polyethyleneimine (PEI25K), which is a cationic polymer and reported to have a remarkable hemolytic effect (Reul et al., 2009). Pegylated NPs

28 and non-pegylated NPs were found to be non-hemolytic (<5% hemolysis), whereas PEI25K exhibited significant hemolysis in order of 39%, suggesting that the prepared NPs were devoid of any unfavorable interaction with red blood cells (Fig. 37). Figure 21: Hemolysis of red blood cells after incubation with pegylated, non-pegylated NPs and gemcitabine. Data reported as mean of three independent experiments Protein adsorption behavior Most of the intravenously administered colloidal delivery systems with hydrophobic surfaces are generally opsonized by plasma proteins, followed by macrophages uptake. The recognition and uptake of NPs by the macrophage took place through cellular receptors specific for plasma proteins bound to the carriers, rather than the carriers themselves (Mosqueira et al., 2001). However, the PEG molecules on the surface of NPs provide a shielding effect by preventing the interaction with serum proteins (Barratt, 2000). In order to have an insight into protein adsorption characteristics, the non-pegylated and pegylated NPs were incubated with HSA solution and the amount of HSA adsorbed on NPs was quantified. The amount of HSA adsorbed on non-pegylated NPs and pegylated NPs was 63±7µg/mg and 36±4µg/mg respectively, suggesting that non-pegylated NPs were more susceptible to opsonization as compared to pegylated NPs. Non-pegylated NPs with hydrophobic surface favor protein adsorption, however, hydrophilic PEG moiety onto the surface of the pegylated NPs reduces the tendency for protein adsorption because it sterically avoids the interaction with proteins Quantitative macrophage uptake After intravenous administration of the particulate delivery system, factors responsible for rapid clearance of NPs from the blood circulation includes hydrophobic exterior (Lee et al., 2011),

29 positive charge (Dong and Feng, 2004) and large diameter (Gaumet et al., 2008). Particles with these characteristics have more affinity to the opsonic proteins, which leads to high macrophage uptake and resulted in rapid clearance from blood circulation. Quantitative macrophage uptake study of non-pegylated and pegylated NPs was executed to investigate the degree of association with macrophages in order to investigate the long blood circulatory property of NPs. Two macrophage cell lines (J774A and THP-1) were used to evaluate this behavior. The relative uptake of C-6 loaded non-pegylated and pegylated NPs after 4h of incubation at 37 0 C in macrophage cell lines is shown in Fig. 38. The results revealed that the uptake efficiency of nonpegylated NPs in the selected macrophage cells was in the order of 44% while the pegylated NPs exhibited approximately 3% macrophage uptake in both the cell lines. The results indicated the potential of pegylated NPs in conferring the circulation longevity. Moreover, it was observed that NPs with 10% of PEG content in copolymer were optimum for long-circulating characteristics (Sheng et al., 2009). In our study as well, pegylated NPs the diminished uptake by macrophages owing to the reduced opsonization could be because of the availability of PEG on the particle surface at an optimum concentration. Figure 22: Uptake of pegylated and non-pegylated NPs by macrophage cell lines (THP-1 and J774A). C-6 loaded 100µl of NPs (200µg/ml) were incubated with macrophages for 1hr at 37 0 C. The experiment was terminated by washing the cells with PBS 3-4 times and cells were lysed by using 10% Triton X solution. Quantitative measurement fluorescence was performed using fluorescence plate reader to obtained % of NPs internalized by macrophages. The uptake of NPs was expressed as the percentage of fluorescent intensity associated with cells in comparison with the total fluorescence intensity in the feed solution.

30 4.6.6 In vitro cytotoxicity The in-vitro anti-proliferative activity of pegylated and non-pegylated NPs was investigated in MiaPaCa-2 and MCF-7 cell lines. After 48 h of incubation gemcitabine and gemcitabine loaded non-pegylated NPs and pegylated NPs showed the IC50 values as 0.047, and µm respectively in MiaPaCa-2 cell lines. While the order of IC50 values in MCF-7 cell lines for gemcitabine and gemcitabine loaded non-pegylated and pegylated NPs were 3.265, and µm respectively. It was observed that as compared to native gemcitabine, pegylated and non-pegylated NPs exhibited remarkably high cytotoxicity in both the cell lines. Further, there was no significant difference in the cytotoxicity of pegylated and non-pegylated NPs in the selected cell lines. The increased cytotoxicity of the NPs could be as a result of intracellular uptake of NPs via endocytosis while the native gemcitabine depends on nucleoside transporters available on the cell walls to permeate inside the cells and these transporters are found to be reduced in cancer cells (Mackey et al., 1998b). In addition, no significant change in the anticancer activity was observed with the cells exposed to non-pegylated and pegylated NPs. Further, gemcitabine and developed NPs exhibited a greater cytotoxic effect in MIAPaCa-2 cells as compared to MCF-7 cells. This could be attributable to the high sensitivity of MIAPaCa-2 cells to the cytotoxic effects of gemcitabine (Tada et al., 2008) Qualitative cellular uptake In this study, C-6 was used as a fluorescent marker for non-pegylated and pegylated NPs to identify the uptake of NPs in cancerous cells (MiaPaCa-2 and MCF-7). C-6 was encapsulated in the NPs as a fluorescent probe because its hydrophobic nature does not allow leaching it out during the study (Chitkara et al., 2014). Fig. 39 shows the uptake of pegylated and non-pegylated NPs after 3h of incubation with MiaPaCa-2 and MCF-7 cell lines, the green fluorescence was caused by internalized C-6 loaded NPs by the cells. In order to confirm that fluorescence was present inside the cells, the nucleus was counterstained with a blue fluorescent dye (DAPI). The green fluorescence adjacent to the nucleus confirming that NPs were within the cells instead of attached the cell surface. Moreover, nearly all the cells treated with either non-pegylated or pegylated NPs were stained with green fluorescence suggesting that there was no remarkable difference in the cellular uptake behavior of NPs in the selected cell lines.

31 Figure 39: Cellular uptake of non-pegylated and pegylated NPs by fluorescence microscopy against MiaPaCa-2 (A-C) and MCF-7 (D-F) cell lines. NPs uptake by cells showing green fluorescence and nuclei stained with DAPI showing blue fluorescence. Cells were incubated for 3h, washed, fixed and permeabilized and nucleus was stained with DAPI. Images were captured using EVOS Floid cell imaging station Pharmacokinetic investigation The native gemcitabine when administered through intravenous route rapidly cleared from the bloodstream. Nanocarriers can help to amend the pharmacokinetic and biodistribution profile of the drugs. We have evaluated the pharmacokinetic profile of native gemcitabine and gemcitabine encapsulated nanocarrier systems after intravenous administration. It was observed that the half-life (t1/2) of native gemcitabine was 0.2 h while that of non-pegylated NPs was increased to 0.4 h. Instead, there was a noteworthy increase in the t1/2 of pegylated NPs (3.8h). These findings were further supported by other pharmacokinetic parameters, i.e. the total body clearance of gemcitabine was 20.7 L/h/Kg, whereas pegylated and non-pegylated NPs exhibited clearance in order of 6.4 L/h/Kg and 9.54L/h/Kg, respectively (Table 9, Fig. 40). As a result,

32 there was an increase in the systemic exposure (AUC0- ) of pegylated and non-pegylated NPs. The AUC0- for gemcitabine, non-pegylated NPs and pegylated NPs was found to be 96.6, and 312.5ng.h/ml respectively. The recorded pharmacokinetic parameters suggested the usefulness of pegylated NPs as a long circulatory delivery system for gemcitabine. The higher t1/2 of the gemcitabine in blood circulation from pegylated NPs could be due to stealth properties owing to the hydrophilic surface, which was supported by other observations as well i.e. low macrophage uptake and lower tendency for serum protein adsorption. Further, the pharmacokinetic study demonstrated that after intravenous administration, the AUC of the gemcitabine loaded pegylated NPs was about three times higher than that of native gemcitabine. Whereas, gemcitabine loaded non-pegylated NPs showed around two times higher AUC value than native gemcitabine. The drug encapsulated within the particles can be protected from the cytidine deaminase enzyme present in plasma, which converts gemcitabine to its inactive metabolite (Khare et al., 2014). Since, the NPs released the drug in a controlled manner, therefore it could be protected from the degradation in the systemic circulation. Table 5: Pharmacokinetic parameters of gemcitabine, non-pegylated NPs, and pegylated NPs Parameters Gemcitabine Non-pegylated NP Pegylated NP AUC(0- ) ng.h/ml Cl (ml/h/kg) t1/2 (h) Figure 40: Pharmacokinetic of gemcitabine, gemcitabine loaded pegylated NPs and nonpegylated NPs in mice following intravenous administration via tail vein at a dose of 2mk/kg. Data are shown as mean ± S.D (n=4).

33 4.6.9 In vivo antitumor efficacy Encouraged by the improved pharmacokinetic of gemcitabine loaded pegylated NPs, the in vivo efficacy of pegylated and non-pegylated NPs was determined by measuring the tumor growth inhibition of Ehrlich ascites based solid tumor bearing Balb-c mice. The control group exhibited a rapid growth in tumor with tumor volume in the order of about 1250 mm 3 at day 13. After administration of three doses of gemcitabine, the tumor volume in the mice was approximately 676 mm 3. While the tumor volume in the group treated with non-pegylated and pegylated NPs was 622 mm 3 and 436 mm 3, respectively (Fig. 41A). The increased efficacy of pegylated NPs could be attributed to long circulatory potential, which in turn lead to the passive targeting of nanocarrier in the tumor tissue. Further, the change in mean body weight of all the treated groups was insignificant as compared to the control group (Fig. 41B). In addition, no serious adverse effect, indicated by changes in body weight, was observed in any of the treated group.

34 Figure 41: In-vivo anticancer activity of gemcitabine and gemcitabine loaded pegylated and non-pegylated NPs. Mice with experimentally induced tumor were treated with three equivalent doses (2mg/kg) of gemcitabine and gemcitabine loaded NPs administered via the tail vein. At the end of the experiment, animals were sacrificed and anti-tumor activity was determined. (A) Image showing growth of average tumor volume after three consecutive doses of gemcitabine and gemcitabine loaded formulation in comparison with control. (B) Graphical representation of the change in body weight at different time intervals after treatment with gemcitabine and gemcitabine loaded NPs in comparison with the control (normal saline). 4.7 Synthesis and Characterization of PLGA-PEG-Folate FT-IR The FT-IR spectra of PLGA, PLGA-PEG bis-amine and PLGA-PEG-Folate is shown in Fig. 42. In the FT-IR spectra of pure PLGA, the peak at 1753 cm -1 is exhibited due to the absorbance of the carbonyl group in PLGA matrix. Also in IR-spectrum of PLGA peaks attributed at 2998 cm -1, 2953 cm -1 and 2853 cm -1 corresponds to (C-H) bending vibrations. The conjugation between PLGA and PEG bis-amine was confirmed by the presence of all the characteristic peaks of both polymers including carbonyl group at 1759 cm -1, 2920 cm -1 (C-H stretching) and 1095 cm -1 (CH2-O-CH2) as well as showed the characteristic peak of amine at 3488 cm -1. Further, the IR spectra of PLGA-PEG-Folate showed characteristic peaks of PLGA at 1758 cm -1 attributed to the carbonyl group in PLGA. IR-spectrum showed the characteristic peaks of PEG at 1105 cm -1 due to the presence of (CH2-O-CH2) and characteristic peaks of an amide bond between PEG and folic acid attributed at 1625 cm -1 confirming the formation of PLGA-PEG-Folate conjugate. The IR spectra also showed characteristic peaks of folic acid including the band at 1488 cm 1 attributed to the characteristic absorption band of the phenyl ring, whereas the one at 1404 cm 1 corresponds to O-H deformation band of the phenyl skeleton.

35 Figure 232: Fourier transforms infrared spectra of PLGA, PEG bis-amine and mpeg-plga- Folate scanned in the region of 4000 to 500 cm -1 using chloroform as solvent H NMR The 1 H NMR spectra of PLGA, PLGA-PEG bis-amine and PLGA-PEG-Folate is shown in Fig. 43. The recorded 1 H NMR spectra of pure PLGA showed the typical signals approximately 1.48 ppm, attributed to the methyl groups of the D-and L-lactic acid monomers, and the multiplets around 5.23 and 4.92 ppm, due to the CH groups of lactic acid and CH2 groups of glycolic acid, respectively. Whereas PLGA-PEG-amine copolymer showed characteristics peak of PLGA and singlet peak at 3.7 ppm (CH2-CH2-O)m observed as the characteristic peak of PEG confirm the formation of PEG-PLGA conjugate. The NMR studies of PLGA-PEG-Folate conjugate showed characteristic peaks of PLGA and PEG as described above and peaks at 6.6, 7.0, 7.3 and 7.8 represents the protons associated with folic acid. The amount of folic acid content determined in the synthesized was 15.21±1.06 µg/mg of the copolymer.

36 Figure 43: Proton NMR (400 MHz) spectra of PLGA, PLGA-PEG-bisamine, and PLGA-PEG- Folate in CDCl3, recorded using trimethyl silane as an internal standard Characterization of PLGA-PEG-Folate NPs Size, zeta potential, and drug loading The loading amount, average particle size and zeta potential of PLGA-PEG-Folate NPs was given in the Table-10. The loading efficiency was optimized using varying the drug/copolymer ratio. The highest loading was obtained in the NPs prepared using drug/copolymer ratio 1:5 and 1:7. However, there was no significant difference obtained in the loading efficiency of these NPs. Therefore, formulation prepared using 1:7 drug/copolymer was considered as optimized formulation and all further investigations was performed using the same formulation. Zeta potential is an imperative feature for the stability of the NPs suspension. The zeta potential was found in a negative range in all the formulations prepared, indicating high electric charge on the exterior of the PLGA-PEG-Folate NPs, which can create high repellent forces between particles to limit aggregation of the NPs in buffer solution (Ebrahimnejad et al., 2009).

37 Nanoparticle Table 6: Characteristics of PLGA-PEG-Folate NPs Drug- Copolymer Ratio Size (nm) PDI Zeta Potential (mv) Loading (µg/mg) PLGA-PEG-Fol 1:5 288 ± ± ± ± 0.23 PLGA-PEG-Fol 1:7 267 ± ± ± ± 1.6 PLGA-PEG-Fol ± ± ± ± 0.82 PLGA-PEG-Fol 1: ± ± ± ± Morphological investigation of PLGA-PEG-Folate NPs The morphology of the PLGA-PEG-Folate NPs was examined by using TEM and SEM as shown in Fig. 44 B and C respectively. The SEM and TEM analysis revealed that the NPs have regular and spherical shape and bear a smooth surface morphology and nanometric range. Both the microscopic techniques suggested that functionalized NPs of PLGA-PEG had an average diameter of about 220 nm. However, these results showed a significant difference in the size obtained by DLS. The different in the size obtained DLS either with TEM or SEM may be due to hydration layer of particles measured using DLS. However, the size particles measured under either SEM or TEM were completely dry and there was no hydration layer present In-vitro drug release studies The in-vitro release profile of gemcitabine from PLGA-PEG-Folate NPs for 72 h is shown in Fig. 44D. The initial burst release was observed in NP formulations during first two hours being greater than 16 %. After this burst release, a constant slow release of the loaded drug amount was observed after 24, 48 and 72 h, showing a typical sustained release of 67%, 73%, and 80% respectively. The release profile demonstrated prolonged drug release of gemcitabine from the nanoparticulate carrier.

38 Figure 244: (A) A representative image of particle size of PLGA-PEG-Folate NPs, (B) TEM image of PLGA-PEG-Folate NPs, (C) SEM image of PLGA-PEG-Folate NPs, (D) Drug release pattern of PLGA-PEG-Folate NPs in PBS buffer at ph Hemolysis The gemcitabine loaded PLGA-PEG-Folate NPs were evaluated in vitro for hemolytic activity against red blood cells. Hemolysis was assessed by determining the amount the hemoglobin release subsequent to the incubation with PLGA-PEG-Folate NPs. The NPs at the concentration of 2mg/kg with respect to gemcitabine concentration was used in comparison with copolymer polyethyleneimine (PEI25K), which is a cationic polymer and reported to have a remarkable hemolytic effect (Reul et al., 2009). PLGA-PEG-Folate NPs showed non-hemolytic activity with less than 5% of hemolysis, whereas PEI25K exhibited significant hemolysis in order of 40%, suggesting that the prepared NPs were devoid of any unfavorable interaction with red blood cells (Fig. 45).

39 Figure 45 : Hemolysis of red blood cells after incubation with PLGA-PEG-Folate NPs and PEI. Triton and PBS were taken as positive and negative control respectively Antiproliferative activity of NPs MTT assay was performed to determine the in-vitro anti-proliferative activity of gemcitabine loaded PLGA-PEG-For NPs. A human breast cancer cell lines MCF-7 was used to evaluate the anti-proliferative activity. The IC50 value of PLGA-PEG-Folate NPs obtained after 48h of incubation showed that gemcitabine encapsulated in the nanoparticulate formulation was significantly more cytotoxic as compared to native gemcitabine. The IC50 value of native gemcitabine obtained was µm whereas the IC50 value of gemcitabine loaded PLGA-PEG- Folate NPs was µm when incubated with MCF-7. The increase in cytotoxicity of PLGA- PEG-Folate nanoparticle could be due to ligand-mediated uptake into the cells Qualitative cellular uptake The qualitative cellular uptake study was performed using C-6 loaded PLGA-PEG-Folate NPs and PLGA NPs in MCF-7 cell lines. Fig. 46 shows the uptake of PLGA NPs (A-C) and PLGA-PEG- Folate NPs (D-F) after 3h of incubation with MCF-7 cells, the green fluorescence was due to C-6 loaded NPs internalized by the cells. In order to confirm that fluorescence was present inside the cells, the nucleus was counterstained with a blue fluorescent dye (DAPI). The green fluorescence adjacent to the nucleus confirming that NPs were within the cells instead of attached the cell surface. Moreover, green fluorescence in the cytoplasm of the cells can be observed in the treatments of either PLGA NPs or PLGA-PEG-Folate NPs. However, the strong fluorescence intensity of folate-targeted NPs was due to active folate receptor-mediated cellular uptake.

40 Figure 256: Cellular uptake of NPs by fluorescence microscopy against MCF-7 cell lines using EVOS Floid cell imaging station: (A-C) PLGA NPs, (D-F) PLGA-PEG-Folate NPs. NPs uptake by cells showing green fluorescence and nuclei stained with DAPI showing blue fluorescence In vivo antitumor effect Gemcitabine loaded PLGA-PEG-Folate and PLGA NPs were evaluated for in vivo antitumor efficacy using the tumor growth inhibition of Ehrlich ascites based solid tumor bearing Balb-c mice. The tumor growth in the saline group was rapid with mean tumor volume expanding up to 1236 ± 12 mm 3 after 13 days (Fig. 47A). Gemcitabine, PLGA NPs, and PLGA-PEG-Folate NPs showed considerable tumor inhibition. Based on the data obtained, the tumor size of the mice followed an order, control (saline) > gemcitabine > PLGA NP >PLGA-PEG-Folate NPs (Fig. 47B). After administration of three doses of gemcitabine, the tumor volume in the mice was approximately 680 ± 38 mm 3. While the tumor volume in the group treated with PLGA NPs and PLGA-PEG-Folate NPs was 622 ± 62 mm 3 and 345 ± 54 mm 3, respectively (Fig. 47A). Results demonstrated the very significant tumor growth inhibition activity of folate-targeted pegylated NPs which is attributed to folic acid mediated active tumor targeting.

41 Figure 267: In-vivo anticancer activity of gemcitabine and gemcitabine loaded PLGA and PLGA- PEG-Folate NPs. Mice with experimentally induced tumor were treated with three equivalent doses (2mg/kg) of gemcitabine and gemcitabine loaded NPs administered via the tail vein. At the end of the experiment, animals were sacrificed and anti-tumor activity was determined. (A) Image showing growth of average tumor volume after three consecutive doses of gemcitabine and gemcitabine loaded formulation in comparison with control. (B) Representative image of tumor isolated from various groups after termination of the experiment.