Article pubs.acs.org/Macromolecules
Amphiphilic Homopolymer Vesicles as Unique Nano-Carriers for Cancer Therapy Shivshankar R. Mane,† Vijayakameswara Rao N,† Koushik Chaterjee,† Himadri Dinda,† Soma Nag,‡ Abhinoy Kishore,‡ Jayasri Das Sarma,*,‡ and Raja Shunmugam*,† †
Polymer Research Centre, Department of Chemical Sciences, and ‡Department of Biological Sciences, Indian Institute of Science Education and Research Kolkata (IISER K), Kolkata, India S Supporting Information *
ABSTRACT: A unique polymersome from amphiphilic, norbornene-derived thiobarbiturate homopolymers (NDTH) and its application as nanocarrier for cancer therapy are elaborately discussed. Various experiments like structural characterizations, control studies, cell viability studies, encapsulation studies, and MTT assay against 4T cancer cells are performed on these NDTH polymersomes to substantiate our claims. All of these results demonstrate that these self-assembled NDTH vesicles have great scope in the world of medicine, and they also symbolize promising carriers for the stimuli-triggered intracellular delivery of hydrophobic drugs.
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INTRODUCTION The field of polymer vesicles (polymersomes) has the phenomenal record of consistent development over the last ten years.1−3 The ability of amphiphilic block copolymers to self-assemble in selective solvents has been widely studied.4 The self-assembled polymersomes are at the forefront of this nanotechnological revolution.5−10 The current research theme of soft-nanotechnology is using polymersomes in the medical applications as nontoxic and targeted drug-delivery agents.11 Though several types of nanocarriers have been proposed for biomedical purposes,12,13 polymersomes (structures similar to lipid vesicles) represent an excellent candidate for medical applications.14−16 These structures are more stable than liposomes but retain their low immunogenicity. But, we are here very specific among the polymersomes formed by the self-assembly of amphiphilic homopolymers,17−21 for their fundamental perspectives along with their potential applications in drug delivery, nanotechnology and as model systems of biomembranes.22−27 Self-assembly due to the strong hydrogen bonding nature, remains a subject of interest in the field of supramolecular chemistry.28 Pioneering work in recognition-induced polymersomes (RIPs) are well-known in the literature. These RIPs are spontaneously formed from a threepoint hydrogen bonding recognition dyads. However, these recognition sensitive structures cannot be used in biomedical applications, due to the complex synthesis and the use of nonpolar media. Living ring-opening metathesis polymerization (ROMP) is more attractive due to the exceptional functional group tolerance of the Grubbs’ catalyst employed in the polymerization process.29−37 Here we have come up with a pH- and lipidsensitive polymersomes from a new molecular architecture, an amphiphilic, norbornene-derived thiobarbiturate homopolymers, © XXXX American Chemical Society
NDTH. On the basis of the hydrophobicity and hydrophilicity of the solvent, the molecular orientation of NDTH is systematically modified. The role of hydrophilic headgroup is enacted by the thiobarbiturate functionality of each monomer unit in NDTH while the norbornene backbone behaves as a hydrophobic moiety. Polymersomes formed by the hydrophilic thiobarbiturate head groups attached to each repeating unit of the hydrophobic norbornene backbone will have greater stability and also are capable of spontaneously responding to their environmental conditions, such as lipophilicity and pH. Cell viability studies on NDTH vesicles suggest the biocompatible nature of these vesicles. The drug release studies in acidic and lipophilic environment demonstrate the stimuli-responsive nature of the novel systems. It is also observed that the drug release from polymersomes of NDTH is significantly accelerated at mildly acid pH of 5.5−6 compared to physiological pH of 7.4, suggesting the pH-responsive feature of the NDTH vesicles. Confocal laser scanning microscopy (CLSM) measurements indicate that these DOXY loaded NDTH vesicles (NDTH-DOXY) are easily internalized by living cells. MTT assay against 4T cancer cells shows that NDTH-DOXY vesicles have a high anticancer efficacy. To our knowledge, this is the first example of a polymersomes formed from an amphiphilic norbornene-derived thiobarbiturate homopolymers employed in the cancer therapy. It does not require motifs like poly(ethylene glycol) (PEG) additionally to make the carrier water-soluble and biocompatible since NDTH itself is water-soluble and biocompatible. Received: August 4, 2012 Revised: September 12, 2012
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Scheme 1. Cartoon Representation of NDTH Polymersomes and Its Stimuli Responsive Drug Release
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the peripheral of the NDTH vesicles. It was observed that a complete disappearance of DOXY emission at 552 and 587 nm took place when the mixture was dialyzed fourth time. After confirming the complete disappearance of the DOXY emission again, the reaction was dialyzed a fifth time. The drug loading efficiency (DLE) was calculated according to the following formula:
EXPERIMENTAL SECTION
Materials. All reagents vinyl ethyl ether and second generation Grubbs’ catalyst (G2) were purchased from Sigma-Aldrich and were used as received without further purification. The solvents dichloromethane (DCM) and methanol were dried over calcium hydride (CaH2) and used for reactions. The deturated solvent dimethyl sulfoxide-d (DMSO-d6) was purchased from Chembridge Isotope Laboratories. Cell Studies. 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) was purchased from USB (Cleveland, OH). Penicillin, streptomycin, and fetal bovine serum (FBS) were purchased from Invitrogen. Vectashield mounting medium with DAPI was obtained from Vector Laboratories. Synthetic Procedure for NDTH. For the synthesis of NDTH,29 a 50 mg sample of NDT (see Supporting Information) was weighed into a separate Schlenk flask, placed under an atmosphere of nitrogen, and dissolved in methanol and anhydrous dichloromethane (9:1 v/v %). Into another Schlenk flask, a desired amount of second generation Grubbs’ catalyst 1.06 mg (100 mol %) was added, flushed with nitrogen, and dissolved in minimum amount of anhydrous dichloromethane. Both two flasks were degassed three times by freeze−pump− thaw cycles. The NDT was transferred to the flask containing the catalyst via a cannula. The reaction was allowed to stir at roomtemperature until the polymerization was complete (24 h) before it was quenched with ethyl vinyl ether (0.2 mL). An aliquot was taken for GPC analysis, and the remaining product was precipitated from pentane, dissolved it again THF, passed it through neutral alumina to remove the catalyst and precipitated again from pentane to get a pure homopolymer. Gel permeation chromatography was done in tetrahydrofuran (flow rate =1 mL/min). The molecular weight of polymer using was measured using polystyrene standards. Mn = 38500, PDI = 1.04. 1H NMR (500 MHz, DMSO-d6): δ 8.23(s, 2H), 7.49 (s, 2H), 7.19 (s, 2H), 7.14 (s, 1H), 5.4 (s, 2 H), 4.9 (s, 2H), 3.0(s, 2H). Doxorubicin (DOXY) Encapsulation and Release Studies. The encapsulation, 1 mg of NDTH and 1 mg of doxorubicin (DOXY) in its salt form, were dissolved in 1 mL of water separately and the two solutions were mixed in a vial and stirred for 30 min. Then the mixture was loaded in a dialysis tube (3500 Da cutoff) and dialyzed against 100 mL of water five times. An aliquot of the sample was removed each time and its fluorescence emission at 552 and 587 nm was measured as an indication of the release of nonencapsulated DOXY in
DLE (%) = (mass of drug loaded in vesicles /mass of drug loaded vesicles) × 100% After this drug encapsulation process the drug release study was carried out at room temperature by dialysis method against 50 mL of buffer solution of different pH’s with different time intervals. An aliquot of the sample was removed and the fluorescence emission measured at 552 and 587 nm (Ex = 510 nm). Cell Culture. 4T cells, from a mouse mammary gland cancer cell line, were maintained in RPMI with containing 10% fetal bovine serum (FBS) with penicillin (100 U/mL) and streptomycin (100 μg/mL). The 4T cells were grown for a month's time with 10 passages and maintained in 5% CO2 at 37 °C. Cellular Uptake Study. The cellular uptake behavior and the intracellular distribution of free DOXY and NDTH-DOXY were analyzed using confocal laser scanning microscope (Zeiss, LSM 710). A confluent monolayer of 4T cells were seeded on 12 mm coverslips plated on 24 well tissue culture dishes in the medium. This confluent monolayer cells were treated with either free DOXY or NDTH-DOXY at different concentrations (1 μg to 1 mg) for 24 h. After 24 h of post treatment cells were briefly washed in PBS with Ca2+ and with Mg2+ and fixed with paraformaldehyde (PFA) (4%) for 10 min at room temperature and successively washed with PBS without Ca2+ and Mg2+ and mounted in Vectashiled with DAPI. Cytotoxicity Assay of NDTH-DOXY. The relative cytotoxicity of NDTH-DOXY in 4T cell lines were quantitatively determined using MTT assay. All cell lines were seeded into 96 well plates at 1 × 104 cells/well and maintained in culture for 24 h at 37 °C in the medium. After 24 h medium was removed, cells were washed with PBS and medium containing different concentration of NDTH-DOXY (1 μg-1 mg) were added to the designated wells. The whole experimental plate was incubated for 72 h. A fresh 20 μL sample of MTT from 5 mg/mL B
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400 μg/mL. After measuring the CAC, the NDTH in water solution was characterized by dynamic light scattering (DLS). This NDTH self-assembled (Scheme 1) into polymersomes with average diameter of approximately 96 nm in water. Figure 1a
stock solution was added to each well, followed by incubation for 4 h at 37 °C. After 4 h, medium from the wells were removed and 100 μL of DMSO were added to each well and incubated for 15 min, and the absorbance of the resulting solution was measured at 515 nm. The cell survivals were determined by comparison of optical density with untreated respective control cell cultures. Cell-Growth Inhibition Assay by Trypan Blue Exclusion Method. For the inhibition activities of cell growth and division of NDTHDOXY were quantitatively determined by visual cell counting using a hemocytometer chamber. Cells were seeded at 1.25 × 104 cells in 24 well tissue culture plates. After 24 h of cell plating, NDTH-DOXY of different concentrations (1 μg to 1 mg) was added to the respective wells. Designated wells for control were maintained with respective medium without adding any NDTH-DOXY. Whole plate was incubated for 72 h. The cell counting was performed at different time intervals, 24, 48, and 72 h, by the trypan blue exclusion method. The viable cell population counts were used to determine the extent of cell inhibition and compared with untreated control cell culture for each time point. Characterization. Gel Permeation Chromatography (GPC). Molecular weights and PDIs were measured by Waters gel permeation chromatography in THF relative to PMMA standards on systems equipped with Waters Model 515 HPLC pump and Waters Model 2414 Refractive Index Detector at 35 °C with a flow rate of 1 mL/min. HRMS analyses were performed with Q-TOF YA263 high resolution (Waters Corporation) instruments by +ve mode electrospray ionization. Fluorometry. Fluorescence emission spectra were recorded on a Fluorescence spectrometer (Horiba Jobin Yvon, Fluoromax-3, Xe150 W, 250−900 nm). Nuclear Magnetic Resonance (NMR). The 1H NMR spectroscopy was carried out on a Bruker 500 MHz spectrometer using DMSO-d6, D2O and CDCl3 as a solvent. 1H NMR spectra of solutions in DMSO-d6, D2O and CDCl3 were calibrated to tetramethylsilane as internal standard (δH 0.00). Fourier Transform Infra Red (FT-IR). FT-IR spectra were obtained on FT-IR Perkin-Elmer spectrometer at a nominal resolution of 2 cm−1. Ultra Violet (UV) Spectroscopy. UV−visible absorption measurements were carried out on U-4100 spectrophotometer HITACHI UV−vis spectrometer, with a scan rate of 500 nm/min. Dynamic Light Scattering (DLS). Particle size of QDs were measured by dynamic light scattering (DLS), using a Malvern Zetasizer Nano equipped with a 4.0 mW He−Ne laser operating at λ = 633 nm. All samples were measured in aqueous as well as methanol at room temperature and a scattering angle of 173°. Transmission Electron Microscopy (TEM). Low resolution transmission electron microscopy (TEM) was performed on a JEOL 200 CX microscope. TEM grids were purchased from Ted Pella, Inc. and consisted of 3−4 nm amorphous carbon film supported on a 400mesh copper grid. Atomic Force Microscopy (AFM). The morphology of the polymer was investigated from NT-MDT micro-40 AFM instrument using a semicontact mode at a scan rate of 1 Hz. Scanning Electron Microscopy (SEM). High resolution SEM was performed on a Zeiss microscope. Confocal Laser Scanning Microscopy (CLSM). Confocal microscope images were taken in LSM 710 with microscope axio observer Z.1, Carl Zeiss.
Figure 1. (a) DLS measurement of NDTH vesicles in aqueous solution. The size of the micelles was about 96 nm with 0.189 PDI. (b) SEM image of the sample used for AFM studies. The inset shows zoomed vesicle size 100 nm. (c) TEM images of NDTH vesicles dipcasted on carbon-coated copper grids show the open-mouth vesicles. The inset shows the proposed self-assembled structure of NDTH in aqueous environment.
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shows the number-average hydrodynamic size (Dh). Light scattering measurements confirmed the presence of polymersomes in solution and provided an average diameter of 96 nm, which was in excellent agreement with the AFM results (Figure S11, Supporting Information). The polymersomes of this amphiphilic homopolymers were studied by atomic force microscopy (AFM), scanning electron microscopy (SEM) and transmission electron microscopy (TEM). SEM images (Figure 1b) also revealed the structures with diameters of around 100 nm. A series of TEM images were collected and a representative image as shown in Figure 1c (stained with RuO4) confirmed that the structures previously observed by AFM and SEM were polymersomes.
RESULTS AND DISCUSSION The detailed synthetic procedure and complete characterization of NDTH were discussed in the Supporting Information (Scheme S1; Figures S1 and S2). The main objective of this work was to demonstrate the stability and drug reservoir capabilities of the novel NDTH amphiphilic polymersomes (Scheme 1) in physiological conditions and also to study their performance as a drug delivery carrier. To confirm the polymersomes nature of NDTH, the following chracterization were carefully done. Critical aggregation concentration (CAC) was measured by using pyrene as an extrinsic probe.38−40 The observed CAC for NDTH was C
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the fluorescence emission measured at 552 and 587 nm (Ex = 510 nm) as an indication of the release of the DOXY. This procedure was repeated every 15 min and the results were observed for 180 min. After 120 min, there was no significant increase in the intensity of fluorescence. Similar procedure was carried out to monitor drug release at pH 4, 5, 6, and 7.4 (Figure 2b, and Figure S3−S6, Supporting Information). Apparently, there was no significant release of the drug (less than 5%) from the NDTH-DOXY at pH 7.4, indicating stability of NDTH-DOXY in physiological condition. The maximum drug release was observed at pH 3 as compared to pH 4, 5, 6, and 7.4. The maximum release at the acidic pH also demonstrates the feasibility of potential delivery of the drug from the NDTH-DOXY polymersomes at macrophage compartments whose pH is in the range of 4.7−5.5. A cartoon representation of the overall drug release process is shown in Scheme 1. Before testing the newly designed nano carriers in the living cells, the biocompatibility of NDTH polymersomes was examined prior to DOXY encapsulation. The cell viability experiment was done in cancer cell lines (4T cell lines). All samples were placed under UV light for 20 min and then they were filtered through 0.22 μm filter, were sterilized before incubating with 4T cell lines. The effect on the cell viability was evaluated by incubating it with the increasing concentration of NDTH (1 μg to 1 mg) up to 72 h, after which the viability of the cell was determined by MTT assay (Figure 3b). The effect of NDTH on cell growth and cell division was observed by
Next, to prove the reservoir capabilities of NDTH polymersomes, the encapsulation of hydrophilic Doxorubicin (DOXY) in its salt form was performed. The total drug loading content and encapsulation efficiency were determined by using UV spectrophotometry. The DOXY loading in NDTH vesicles was 70% w/w. To test the DOXY release from the NDTH polymersomes under the lipophilic environment, the most accepted octanol−water system was chosen.41 DOXY encapsulated NDTH polymersomes (NDTH-DOXY) were taken in a vial along with 1 mL of octanol. An aliquot of the sample was removed from octanol layer and the emission at 552 and 587 nm (Ex = 510 nm) was measured as an indication of the release of DOXY (Figure 2a,
Figure 2. (a) DOXY release profile in the lipophilic environment. Inset: encapsulation (bottom layer) and release studies (upper layer) from NDTH polymersomes. (b) pH dependent DOXY release profile.
inset). This procedure was repeated for every 15 min and results were observed for 720 min. It was observed that after 80 min, there was no significant increase in the intensity of emission (Figure 2a). The results strongly suggested the release of DOXY in lipophilic environments. The pH-responsive drug carriers have attracted great attention and are one of the most studied stimuli-sensitive systems because of the presence of pH variations within the body. For the drug release profile of NDTH-DOXY, pH 7.4 as well as acidic condition was studied, because the normal physiological pH of the human blood is 7.4. For the pH-triggered drug release, 1 mg of NDTH-DOXY was dissolved in 1 mL of distilled water and loaded in a dialysis tube (3500; Dalton cutoff) and dialyzed against 50 mL of buffer solution of pH 3 and stirred gently. An aliquot of the sample was removed and
Figure 3. (a) The effect of NDTH on cell growth was determined by trypan blue exclusion method. (b) Cytotoxicity profile of NDTH in 4T cells. 4T cells were seeded at 1 × 104 cells/well in 96 well plates and maintained in their respective medium and treated with different doses of NDTH (1 μg to 1 mg). D
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incubating the nanocarriers in 4T cell lines. Although very high NDTH concentrations appeared to be toxic, these concentrations were well above the amounts typically used for drug delivery systems (Figure 3a). Next, to address the in vivo elimination kinetics of the newly designed NDTH polymersomes, oral administration of NDTH to 8 weeks old C57BL/6 mice were made following the institutional biosafety and ethical committee guidelines. Urine samples were collected every 1 h and analyzed using fluorescence spectroscopy. Since NDTH was emissive in nature, the emission of NDTH was utilized as a chromophore to monitor the renal clearance of NDTH polymersomes. The solutions were excited at 285 nm and the emission maximum of NDTH was monitored as response at 387 nm (Figures S7−S10, Supporting Information). The signal from NDTH was sustained for more than 36 h after which it fell into the background level as shown in Figure 4b. A cartoon representation of the overall process
Figure 5. CLSM pictures for the uptake of NDTH-DOXY in 4T cells. Confluent monolayer cells were loaded with NDTH-DOXY at different concentrations (5−40 μg) for 24 h.
A mouse mammary gland cancer cell line (4T) was chosen to study the in vitro efficacy of the release of of NDTH-DOXY. The cell line was grown in culture with similar kinetics and the pH of the cell medium. Uptake of NDTH-DOXY in all the cells were through endocytosis. So the uptake occurred relatively slowly inside the cell compare to free DOXY. The uptake of free DOXY occurred in about 6−8 h in all the cell lines which could be clearly visible by fluorescent microscope after 12 h of post treatment (data not shown). Interestingly, the uptake of NDTH-DOXY appeared to be different. Post treatment of NDTH-DOXY at 24 h showed a diffused intracellular accumulation. There was a clear endosomal/lysosomal NDTHDOXY penetration was observed from the confocal laser scanning microscopy (CLSM) as shown in Figure 5. The known acidic environment formed during endosomal uptake process would induce DOXY release from the NDTH-DOXY in 4T cells.
Figure 4. (a) Cartoon representation of the renal clearance of NDTH. (b) A plot of fluorescence intensity vs time for the urine sample collected from the 8 weeks old C57BL/6 mice after oral administration of CP2. The error bars are based on five mice in each group.
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is shown in Figure 4a. The profiles of elimination kinetics indicated that the NDTH polymersomes have longer circulation time inside the body. This is due to the prolonged in vivo circulation time of the NDTH polymersomes. Having addressed the biocompatibility and renal clearance of NDTH polymersomes, in vitro efficacy and cellviability of DOXY loaded NDTH polymersomes (NDTH-DOXY) was explored to demonstrate its potential application in stimuli responsive cancer therapy.
CONCLUSIONS In conclusions, polymersomes obtained from the self-assembly of amphiphilic NDTH homopolymers have been used as a nano reservoir for controlled release of doxorubicin (DOXY), a model anticancer drug. These novel polymersomes are proved to be capable of loading high drug quantities with a reasonable loading efficiency (about 70% w/w). The drug release studies E
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in acidic and lipophilic environment demonstrate the stimuliresponsive nature of the novel systems. CLSM measurements indicate that these DOXY-loaded NDTH vesicles (NDTHDOXY) can be easily internalized by living cells. MTT assay against 4T cancer cells confirms that NDTH-DOXY vesicles have a high anticancer efficacy. These features proclaim a great promise for the development of NDTH polymersomes as viable drug delivery systems. Moreover the advantages are many as they provide: biocompatibility, high loading capability, prolonged in vivo circulation, and sustained drug release that could be accelerated in acidic environments.
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ASSOCIATED CONTENT
S Supporting Information *
Details of synthetic procedure and additional analytical data. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail: (R.S)
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS S.R.M., V.N. and A.K. thank IISER-Kolkata for the research fellowship and R.S., H.D., and K.C. thank the DST, New Delhi for Ramanujan Fellowship. S.N. thanks the CSIR, New Delhi, for the funding. R.S. and J.D.S. thank IISER-Kolkata for the infrastructure and start up funding.
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