Docetaxel-conjugated monomethoxy-poly(ethylene glycol)-b-poly(lactide) (mPEG-PLA) polymeric micelles to enhance the therapeutic efficacy in oral squamous cell carcinoma

Abstract

In this study, docetaxel (DTX) was successfully conjugated to the monomethoxy-poly(ethylene glycol)-b-poly(lactide) (mPEG-PLA) polymer block via an ester linkage (DTX-PM). The polymer–drug conjugate resulted in the formation of nanosized micelles with a clear spherical shape. This size range is suitable for passive targeting via an EPR effect in cancer drug delivery. The DTX-PM exhibited a sustained release of the drug over 160 h with slightly accelerated release at acidic conditions. A cytotoxicity assay clearly revealed a time-dependent anticancer effect of DTX-PM in the squamous cancer cells. At 24 h incubation, the free drug exhibited a potent anticancer effect in the cancer cells while DTX-PM exhibited a superior anticancer effect by the end of 48 h incubation. The higher cytotoxic potential of DTX-PM suggests potential therapeutic advantages for cancer treatments. The annexin-V/PI based apoptosis assay further confirmed the anticancer potential of DTX-PM. The DTX-PM induced significantly higher cancer cell apoptosis in HSC-3 cancer cells. Importantly, DTX-PM significantly controlled the tumor progression in HSC-3 cancer cells bearing a tumor xenograft. Consistently, DTX-PM treated mice however showed a severe necrosis of the cancer cells in H&E staining and showed remarkably higher TUNEL positive apoptotic cells. Overall, a polymer–drug conjugate (DTX-PM) is a promising strategy to improve the therapeutic efficacy in oral squamous cell carcinoma.

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Introduction

Oral squamous cell carcinoma (OSCC) is one of the most common forms of oral cancers responsible for more than a 90% death rate.^1,2 Oral cancer accounts for the sixth most cancer-related incidence worldwide. OSCC is often reported to be associated with poor prognosis and is aggressive and highly metastatic in nature.³ Patients with OSCC are reported to have low or poor survival rate. Despite the advances have been made in diagnosis and therapeutic modalities for OSCC therapy, the morbidity rate of OSCC has not improved significantly and still remains a cause of concern.^4–6 Therefore, new treatment strategies have to be made to effectively treat OSCC with negligible effects on the normal cells and thereby avoiding adverse effects.

In this regard, docetaxel (DTX), a taxane drug is indicated in the first line treatment of oral cancers and moreover it is twice as potent as paclitaxel. DTX binds to microtubules and inhibits microtubule depolymerization, resulting in the disruption of tubule equilibrium system.⁷ This leads to the arrest of G2/M phase of cell-cycle, cell apoptosis and eventual cell death. However, potential benefits of DTX could not be reaped owing to its poor aqueous solubility, non-specific distribution (suffer from poor pharmacokinetics), and systemic side effects in the body.^8,9 Especially, low molecular weight of DTX resulted in short circulation times and with low concentrations in tumors and metastases. There are few formulations available in the market (Taxotere®), however it did not improve the quality of healthcare and therapeutic efficacy beyond an extent and suffered from severe adverse effects.¹⁰ Therefore, efforts have been continuously made to improve the pharmacological effect of DTX in the cancer cells.

Nanotechnology has potential for selectively delivering anti-cancer agents into tumor cells without affecting normal healthy tissues. Many successful strategies have incorporated targeted drug delivery capabilities for nanoparticles.¹¹ Many different delivery systems with desirable properties have been developed time and again to increase its accumulation in the tumor tissues while attenuating their accumulation in potentially endangered healthy organs and tissues.¹² Among the many delivery systems or nanoparticle systems, polymeric micelles which are formed by the self-assembly of amphiphilic block polymers have drawn the great interest of researchers from across globe.^13,14 The polymeric micelles are characterized by a typical core–shell architecture where the core consists of hydrophobic core while shell mostly consists of the polyethylene glycol (PEG) which imparts particle stability in an aqueous solution.¹⁵ The self-assembled polymeric micelles could improve the tumor localization by virtue of Enhanced Permeability and Retention (EPR) effect wherein nanosized particles tend to penetrate the cancer cells with leaky blood vessels with defective lymphatics enabling them to efficiently accumulate in tumors over time.¹⁶ Tumor blood vessels, unlike those occurring in normal tissues, have 600–800 nm wide gaps between adjacent endothelial cells, leading to a defective vascular system with poor lymphatic drainage.¹⁷ The chemotherapeutic effect of paclitaxel (PTX) was shown to increase when administered in the micellar formulations (NK105). A micellar formulation of PTX (Genexol-PM) showed 59% response rate in metastatic breast cancer model. Similarly, CT-2103, a polymer–drug conjugate of paclitaxel and poly-L-glutamic acid were shown to increase the antitumor efficacy in cancer cells. Moreover, novel free poly(L-g-glutamylglutamine) (PGG)-PTX conjugate nanoparticles were developed and showed an effective tumor growth inhibitory effect in a mouse model. Taking all of the above reasoning's into account, we have designed a novel strategy to conjugate DTX to the poly lactic acid (PLA).

In this work, we have reported the synthesis of a novel polymer–drug conjugate, monomethoxy-poly(ethylene glycol)-b-poly(lactide) (MPEG-PLA)-docetaxel in which drug is covalent conjugated with the polymeric block which upon self-assembly will reside in the core of the micelles. The PLA will form the core of the micelles while PEG will form the hydrophilic shell. Owing to the biodegradability of PLA, it can be expected that the polymer–drug conjugate will erode slowly while releasing the DTX in a gradual sustained manner. To prepare mPEG-PLA-DTX, hydroxyl-terminated polymer block mPEG-PLA was synthesized by the polymerization of lactide with stannous octoate as catalyst and PEG as a microinitiator. The hydroxyl group was converted to carboxyl group and then conjugated to docetaxel. The mPEG-PLA based micelles have been reported as one of the advanced carriers owing to the biocompatibility, biodegradability, nontoxicity, low immunogenicity, and good mechanical properties.^18,19

The main aim of present study was to increase the chemotherapeutic potential of docetaxel to treat the oral squamous cell carcinoma (OSCC). For this purpose, DTX was chemically conjugated to the polymeric block and formed the self-assembled micelles. The drug-conjugated micelles were characterized for size, shape and release kinetics. The anticancer effect of drug-loaded micelles was evaluated in squamous cell carcinoma HSC-3 cell line. Importantly, in vivo antitumor efficacy study was performed in xenograft tumor model.

Results

Preparation of polymer–drug conjugated micelles

The mPEG-PLA block copolymer was synthesized by the ring opening polymerization of lactide with the help of microinitiator mPEG. The hydroxyl group of the mPEG-PLA polymer block was functionalized with an active group that can react with the hydroxyl groups of docetaxel. For this purpose, hydroxyl groups were converted to carbonyl group by reacting with succinic anhydride. The carboxyl group terminated mPEG-PLA-COOH was then conjugated with the hydroxyl group of docetaxel (Fig. 1).²⁰ The particle size of polymeric micelles was evaluated using dynamic light scattering method (DLS). The average size of DTX-PM was observed to be around 58.2 ± 2.3 nm with an excellent polydispersity index (Fig. 2a). The morphology of polymeric micelles was evaluated using transmission electron microscope (TEM). The TEM image showed a uniform spherical shaped particles on the copper grid indicating the self-assembly nature of the micelles. The micelles were stably present on the TEM grid without any sign of aggregation (Fig. 2b). Moreover, size measured from TEM was consistent from the size measured from DLS analysis.

Fig. 1 Schematic illustration of synthesis of mPEG-PLA-docetaxel polymer drug conjugate.

Fig. 2 Physicochemical characterization of nanomicelles (a) transmission electron microscope (TEM) image and (b) particle size distribution of DTX-PM.

Drug release study

In vitro drug release patterns are widely used to predict in vivo pharmacokinetics. The in vitro release kinetics of DTX from DTX-PM was investigated under phosphate buffered saline (PBS, pH 7.4) and acetate buffered saline (ABS, pH 5.0) at 37 °C (Fig. 3). The DTX-PM exhibited excellent stability in the PBS medium with less than 25% release at 24 h study period. The release rate was further sustained for rest of study period up to 160 h with approximately 70% drug release. On the other hand, staggered release of ∼35% was observed in ABS conditions after 24 h. The release of DTX in ABS was higher than that in PBS throughout all study period. Almost all the 100% of drug was released by the end of 160 h in ABS conditions.

Fig. 3 The in vitro release of DTX from DTX-PM was carried out by dialysis method. The release study was carried out in phosphate buffered saline (PBS, pH 7.4) and acetate buffered saline (pH 5.0) at 37 °C.

Cytotoxicity assay

To determine the cytotoxicity of free DTX and DTX-PM, cells were treated with the formulations with varying concentrations and incubated for 24 and 48 h, respectively and analyzed using MTT assay. As can be seen (Fig. 4a and b), two distinct trends could be seen at 24 h and 48 h. At 24 h incubation, free drug exhibited a potent anticancer effect in the cancer cells while DTX-PM exhibited superior anticancer effect by the end of 48 h incubation.

Fig. 4 Cell viability of HSC-3 cancer cells after incubation with different formulations for 24 h (a) and 48 h (b), respectively; (c) effect of blank polymer on the cell viability of cancer cells. The cells were treated increasing concentration of formulations and assayed by MTT protocol.

Apoptosis assay

In the present study (Fig. 5), cells were treated with respective formulations for different time point and stained with annexin V/PI combinations. Consistent with the cytotoxicity assay, free drug and DTX-PM showed a similar level of apoptosis (∼20%) after 24 h incubation. The level of apoptosis was significantly different at longer incubation period. The DTX-PM showed nearly 32% of cancer cell apoptosis comparing to that of ∼23% apoptosis by free DTX.

Fig. 5 Cancer cell apoptosis of HSC-3 squamous cancer cell after treatment with free DTX and DTX-PM. The apoptosis was evaluated using annexin-V/PI staining kit and detected by flow cytometer. The study was carried out after 24 and 48 h incubation.

Antitumor efficacy study

The antitumor efficacy of free DTX and DTX-PM was investigated in HSC-3 cancer cell bearing tumor xenograft model (Fig. 6a). The mice were injected with 5 mg kg⁻¹ of respective formulation and the tumor volume was noted at predetermined time intervals. As seen, free DTX did control the progression of tumor; however, it was not prominent. As expected, DTX-PM significantly controlled the tumor burden by great margin and almost 3-fold more effective than the free DTX treated group. The body weight analysis indicates that the nanocarrier effectively controlled the drug-induced systemic toxic effect (Fig. 6b). Free DTX induced a more than 10% lose in body weight while DTX-PM did not show such sign of body weight loss.

Fig. 6 In vivo antitumor efficacy of free DTX and DTX-PM in xenograft tumor model in terms of (a) tumor volume and (b) body weight. The drugs were administered at a fixed dose of 5 mg kg⁻¹ every 3 days for a total of 3 injections. The mean tumor volumes were calculated using a vernier caliper and drawn as a function of time. ***p < 0.0001.

Histopathological and TUNEL assay

H&E staining was performed on the tumor samples to observe the antitumor efficacy of different formulations. As seen (Fig. 7a), nucleus was intact with typical features in untreated mice group. The DTX-PM treated mice however showed a severe necrosis of the cancer cells. In addition, neutrophils and macrophages were also observed in the necrosis area of tumor sections.

Fig. 7 Hematoxylin and eosin staining of tumor samples and evaluation of apoptosis by TUNEL assay in xenograft tumors. The tumors were extracted from mice and fixed in 10% formalin solution and slices were made.

TUNEL assay was further performed to confirm the anticancer effects of different formulations (Fig. 7b). As seen, fewer apoptotic cells were seen in control group while remarkably higher TUNEL positive apoptotic cells were observed in DTX-PM treated groups. All these outcomes conclude the superior anticancer efficacy of polymer–drug conjugates.

Discussion

In this study, we attempted to combine the benefits of anticancer drug along with the nanotechnological solutions. OSCC often reported to have low or poor survival rate and is the most common forms of oral cancers responsible for more than 90% of death rate. Despite the advances have been made in therapeutic modalities for OSCC therapy, the morbidity rate of OSCC did not improve significantly and still remain a cause of concern. Docetaxel (DTX) is indicated in the first line treatment of oral cancers and moreover it is twice as potent as paclitaxel. DTX induce the cancer cell death by inhibiting the microtubule depolymerization and arrests the cells at G2/M phase of cell cycle and results in cell apoptosis. However, clinical outcome of DTX has been hampered by the poor aqueous solubility and systemic side effects in the body. In this study, therefore, we have reported the synthesis of a novel polymer–drug conjugate, monomethoxy-poly(ethylene glycol)-b-poly(lactide) (MPEG-PLA)-docetaxel to increase the therapeutic efficacy in OSCC. This carrier material was selected owing to its potential to increase the blood circulation of DTX in the body.

The mPEG-PLA was reported to be one of the bioactive and biodegradable carriers. The DTX-conjugated PLA-b-PEG copolymers could undergo self-assembly into micelles in aqueous solution, due to the hydrophobic interaction of the PLA segment. The average size of DTX-PM was observed to be around 58.2 ± 2.3 nm with an excellent polydispersity index. It has been reported that the long hydrophobic segment (DTX and PLA) could promote the compactness of the core of the micelles and could result in the formation of smaller nanoparticles. The nanosized particles are suitable for the cancer targeting which can pass through the tumor spaces using EPR effect.

The DTX-PM exhibited excellent stability in the PBS medium with less than 25% release at 24 h study period. These results indicate that drug–polymer conjugate was relatively stable in alkaline conditions while slightly unstable in the acidic conditions. Moreover, based on the release kinetics, it can be expected that the polymer–drug conjugate will minimize the premature drug release during blood circulation and provide sufficient concentration in the cancer cells. The enhanced release of drug in the acidic conditions could increase the accumulation of drug in the tumor tissues and thereby enhanced therapeutic efficacy.

The higher cytotoxic effect of free DTX initially was attributed to immediate diffusion of drug into the cancer cells and readily available for the anticancer effects, while, drug has to detach from the micelles (from polymers) to perform its pharmacological function resulting in less therapeutic effect. On the other hand, at longer incubation, micelles showed more profound cytotoxic effect than that of free drug. This could be attributed to the sustained release of drug from the nanocarriers and kills the cancer cells in a gradual manner. Consistently, DTX-PM showed lower IC50 value than compared to that of free DTX at the end of 48 h. Nevertheless, regardless of high cytotoxic effect of free DTX in in vitro conditions free DTX does not have tumor-targeting abilities in vivo which can lead to severe side effects. The higher cytotoxic potential of DTX-PM suggests the potential therapeutic advantages for in vivo delivery. Our results are in concordance with the previously published reports wherein the nanomicelles showed increased in vitro cytotoxicity toward cancer cells.²¹

Annexin V is a Ca²⁺-dependent phospholipid-binding protein that has high affinity for the phosphatidylserine (PS, a membrane phospholipid). The PS translocate from inner to the outer membrane surface before the integrity of plasma membrane loses. Therefore, viable cells with intact plasma membrane will exclude the propidium iodide while the dead or apoptotic membrane will allow the PI to permeate the cancer cells. Based on these facts, FITC-annexin V and PI staining would provide crucial information about the stages of apoptosis and viable nature. The DTX-PM showed nearly 32% of cancer cell apoptosis comparing to that of ∼23% apoptosis by free DTX. The remarkable apoptosis effect of DTX-PM was attributed to the sustained release nature of micelles which might be coupled with better internalization of nanosystem as reported elsewhere.

As expected, DTX-PM significantly controlled the tumor burden by great margin and almost 3-fold more effective than the free DTX treated group. The results clearly confirm the superior anticancer effects of polymeric micelles conjugated drug over free drugs. The superior anticancer effect of polymeric micelles might be due to the small particle size, long blood circulation time, as well as the passive targeting via EPR effects.^22–24

Conclusion

In conclusion, docetaxel was successfully conjugated to the mPEG-PLA polymer block via an ester linkage. The polymer–drug conjugate resulted in a formation of nanosized micelles with a clear spherical shape. This size range is suitable for passive targeting via EPR effect in cancer drug delivery. The DTX-PM exhibited a sustained release of drug over 160 h with slightly accelerated release at acidic conditions. Cytotoxicity assay clearly revealed a time-dependent anticancer effect of DTX-PM in the squamous cancer cells. At 24 h incubation, free drug exhibited a potent anticancer effect in the cancer cells while DTX-PM exhibited superior anticancer effect by the end of 48 h incubation. The higher cytotoxic potential of DTX-PM suggests the potential therapeutic advantages for cancer treatments. The annexin-V/PI based apoptosis assay further confirmed the anticancer potential of DTX-PM. The DTX-PM induced significantly higher cancer cell apoptosis in HSC-3 cancer cell. Importantly, DTX-PM significantly controlled the tumor progression in HSC-3 cancer cell bearing tumor xenograft. Consistently, DTX-PM treated mice however showed a severe necrosis of the cancer cells in H&E staining and showed remarkably higher TUNEL positive apoptotic cells. Overall, polymer–drug conjugate (DTX-PM) is a promising strategy to improve the therapeutic efficacy in oral squamous cell carcinoma.

Materials and methods

Materials

Stannous octoate (Sn(Oct)₂), dicyclohexylcarbodiimide (DCC), and 4-dimethylamino-pyridine (DMAP) were procured from Sigma-Aldrich (China). Monomethoxy PEG(MPEG) (M_n = 8 kDa) was purchased from Seebio Biotech Inc, China. D,L-Lactide was purchased from Daigang Co. Ltd, China. Docetaxel was obtained from Sigma-Aldrich, China. All other chemicals were of reagent grade and used without further purification.

Synthesis of PEG-b-PLA-COOH

For this, hydroxyl group terminated PEG-PLA was first synthesized. D,L-Lactide was polymerized using mPEG as a linear initiator and Sn(Oct)₂ as a catalyst. In brief, D,L-lactide, mPEG, Sn(Oct)₂ were added to a tube and filled with nitrogen gas and sealed under vacuum. The compounds were polymerized at a high temperature of 130 °C for 18 h. The final product was placed in dichloromethane solution and precipitated by means of anhydrous diethyl ether and vacuum dried. The so-formed PEG-PLA-OH was esterified with succinic anhydride using DMAP and TEA as a catalyst. In brief, PEG-PLA-OH solid product, DMAP, TEA, SA were co-dissolved in chloroform and stirred for 24 h continuously. The organic solvent was removed and dissolved in DCM solution and the product was precipitated by diethyl ether. The solid product was collect by vacuum drying. The carboxyl content of PEG-b-PLA-COOH was determined by non-aqueous titrations using a base such as sodium hydroxide.

Synthesis of PEG-b-PLA-docetaxel

To prepare PEG-PLA-docetaxel, 100 mg of PEG-PLA-COOH and 25 mg of DTX was dissolved in anhydrous methylene chloride. Followed by 5 mg of DCC and 3.5 mg of DCC were added to the above mixture at 0 °C. The organic mixture was stirred for 24 h at 0 °C. After that, precipitate was filtered and filtrate was washed with HCl/water mixture. The organic phase was poured in diethyl ether solution and the final product was isolated, vacuum dried, and stored. The yield was found to be 82%.

Particle size and zeta potential analysis

The particle size and zeta potential was analyzed using dynamic light scattering method (Zeta Sizer, Malvern Instruments, UK). The samples were suitably dilute before the measurements.

Morphological analysis

The morphology of nanoparticles were determined by field emission transmission electron microscope (FE-TEM) (JEM 2100F, JEOL, Japan). The samples were diluted and placed in a copper grid. The samples were blotted out and counterstained with 2% phosphotungstic acid (PTA). The samples were air dried and observed under TEM.

Drug release study

The release of DTX from DTX-PM system was evaluated by dialysis method. The release study was performed at 37 °C in pH 7.4 PBS solutions and pH 5.0 acetate buffer medium to mimic physiological and tumor conditions. The lyophilized samples containing 2 mg equivalent of DTX was incubated in 1 ml of respective release medium and placed in a dialysis bag (MW ∼ 1 kDa). The dialysis bag was placed in a 50 ml of release medium and stirred at a constant speed at 37 °C. At predetermined time, 1 ml of release samples were withdrawn and replaced with equal amount of fresh release medium. The amount of drug released was evaluated by means of HPLC. A Beckman HPLC system consisting of a model 126 pump, 166 detector, and 507 autosampler. An AllTech RP C₁₈ column (4.1 × 300 mm) was used for analysis. The mobile phase consisted of methanol : water (70/30, v/v) at a flow rate of 1 ml min⁻¹. The docetaxel was detected at 229 nm.

In vitro cytotoxicity assay

The cytotoxic effect of free DTX and DTX-PM was evaluated in squamous cell carcinoma HSC-3 cell line. The cells were cultured in DMEM growth media supplemented with heat inactivated 10% fetal calf serum (FCS), 1% penicillin, and 1% streptomycin in a fully humidified incubator containing 5% CO₂ and 95% air. The cytotoxicity assay was performed by MTT assay. The cells at a seeding density of 1 × 10⁴ cells per well was seeded in a 96-well plate and incubated at 37 °C and in 5% CO₂. Next day, cells were treated with free DTX and DTX-PM at different concentrations and incubated for 24 and 48 h, respectively. Cells were then treated with MTT reagent (10 μl per well volume from 5 mg ml⁻¹ solution in PBS) for 4 h at 37 °C. The cells were then treated with 100 μl of DMSO to dissolve the formazan crystals. The optical density of extracted formazan crystal was recorded at 570 nm using a microplate reader. The percentage of residual cell viability was plotted against different concentration on the x-axis.

Apoptosis assay (annexin V/PI staining assay)

The cells at a seeding density of 5 × 10⁵ cells per well was seeded in a 6-well plate and incubated at 37 °C and in 5% CO₂. Cells were treated with free DTX and DTX-PM at a fixed concentration of 2.5 μg ml⁻¹ and incubated for 20 h. The cells were then harvested and washed with PBS. The cells were then treated with 100 μl of binding buffer and stained with 5 μl of annexin-V FITC and 5 μl of PI (BD Biosciences KIT) and incubated at room temperature for 15 min. The cells were then analyzed by flow cytometry (BD LSR II Analyzer), and 10 000 cells were counted.

Antitumor efficacy study

All animal experiments were performed in accordance with the guidelines framed by ‘Institutional Animal Ethics Committee’, Qilu Hospital, Shandong, China. The antitumor efficacy study was performed in HSC-3 cancer cell bearing xenograft model. The cell suspension (2 × 10⁶ cells/100 μL PBS) was subcutaneously injected into the right flank of the nude mice. When tumors became palpable in 10 days, mice were randomly divided into 3 groups with 8 mice in each group; (a) free DTX (5 mg kg⁻¹) and (b) DTX-PM (DTX equivalent concentration of 5 mg kg⁻¹). The samples were administered 3 times of every 3^rd day. Tumor diameters were measured every 3 days, and tumor volume was calculated using the formula: tumor size = (length) × (width)² × 0.52. All the animals were observed after administration, including the general conditions (the activity, energy, hair, feces, behavior pattern, and other clinical signs), body weight, and mortality.

H&E staining and TUNEL assay

To evaluate the histology of tumor samples, tumor sections were fixed in 10% formalin, dehydrated, embedded in rosin and then stained with hematoxylin and eosin. The tumor slices were then visualized using microscope.

For TUNEL assay, thin tumor sections were stained with TUNEL kit which consists of dUTP labeled labeled by digoxigenin and an anti-digoxigenin antibody conjugated with horseradish peroxidase. The TUNEL assay was carried out as instructed by manufacturers standard assay protocol.

Acknowledgements

This research was supported by grants from the National Natural Science Foundation of China (No. 81300964), The China Postdoctoral Science Foundation (No. 2013M531611 and No. 2014T70648), and The Traditional Chinese medicine science and technology development plan of Shandong Province (No. 2013-190).

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