Biodegradable Magnetic Nanocarrier for Stimuli Responsive Drug

Biocompatible nanocarriers conjugated with magnetic nanoparticle, doxorubicin and poly(ethylene oxide) (PEG) motif have been designed ...
0 downloads 0 Views 8MB Size
Download PDF
Article pubs.acs.org/Macromolecules

Biodegradable Magnetic Nanocarrier for Stimuli Responsive Drug Release Mutyala Naidu Ganivada,† Vijayakameswara Rao N,† Himadri Dinda,† Pawan Kumar,† 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, India S Supporting Information *

ABSTRACT: Biocompatible nanocarriers conjugated with magnetic nanoparticle, doxorubicin and poly(ethylene oxide) (PEG) motif have been designed (PVLPEG-PVLDOXI-PCLPHOS) to create a magnetic vector under magnetic field. Acylhydrazine linker is used to release the drug exactly at the mild acidic conditions resembling the pH of the cancerous cells. All the monomers and polymers are characterized carefully by the routine analytical techniques. Thermogravimetric analysis (TGA), FT-IR spectroscopy and scanning electron microscope (SEM) techniques are employed to confirm the anchoring of iron particle (Fe3O4) to the PVLPEG-PVLDOXI-PCL-PHOS. Reservoir capabilities of the newly designed biodegradable nanocarrier are tested by both dynamic light scattering (DLS) and transmission electron microscopy (TEM). Drug release profile from nanocarrier is monitored by fluorimeter. The release profile shows the importance of having the acylhydrazine linker that helps to release the drug at the mild acidic conditions similar to cancerous cells. Confocal laser scanning microscopy (CLSM) and flow cytometry studies on 4T cells indicate that nanocarriers from PVLPEG-PVLDOXIPCL-PHOS polymer are internalized efficiently. It is very interesting to note that the nanocarriers have exhibited both biologically and magnetically targeting abilities toward 4T cells in vitro.

T

inorganic nanoparticles.8 Aliphatic polyesters, with a welldefined architecture, molecular weight, properties, and free of residual catalyst fragments, are a most desirable choice for developing hybrid materials toward biomedical applications.9a−c They can be potentially biodegradable through enzymatic and chemical hydrolysis.10a,b However, due to its hydrophobic nature, poly-ε-caprolactone (PCL) degrades much slower compared to other polyesters. But this property can be altered by appropriate variations in monomer structure, copolymer composition, and reaction parameters. There are number of examples reported on chain-end functionalized aliphatic polyesters by ring-opening polymerization.11a−c However, pendent functionalization of biodegradable PCL polymers are not explored in detail for the stimuli responsive drug delivery applications. Recently we have reported on pH-responsive norbornene derived amphiphilic polymers to demonstrate the stimuli responsive drug release.12a,b Herein, we report an efficient method to prepare a lactone based smart biodegradable nanocarrier (PVLPEG-PVLDOXI-PCL-PHOS-Fe3O4) using ring-opening polymerization (ROP). In the present work, we have designed unique copolymers having poly-

herapeutic drugs, once administrated, encounter a series of transport barriers, namely, systemic barriers and cellular barriers before they can start functioning at the tumor site.1a−c Because of this, conventional small molecule therapeutic drugs exhibit poor pharmacokinetic profiles, and unsatisfactory therapeutic efficacy. The application of polymeric nanocarriers to oncology explores the use of macromolecules to enhance delivery of therapeutic agents.1a−c The improved efficacy and decreased toxicity are the desired outcomes of using nanocarriers.2a−d Polymeric nanocarriers, due to the enhanced permeability and retention (EPR) effect, provide increased drug solubility, extended drug half-life, and effective targeting to solid tumors.3a−e Though there has been number of successful stimuli-responsive block copolymer based approaches in nanomedicine,4a−d there are only few Food and Drug Administration (FDA) approved examples available for the actual treatment. This is because, in most of the systems, the percent of drug containing nanocarriers (dose) that reaches the tumor still remains low.5a,b So intricate approach and novel strategies are warranted in delivering the cancer drugs.6 This can be achieved by exploring the diverse self-assembled structures and shapes with great drug loading capacity, which can be in blood circulation for a longer time.7a−c Long blood circulation due to desirable shape can be achieved by making hybrid materials of block copolymers and © 2014 American Chemical Society

Received: February 20, 2014 Revised: March 27, 2014 Published: April 3, 2014 2703

dx.doi.org/10.1021/ma500384m | Macromolecules 2014, 47, 2703−2711

Macromolecules

Article

Scheme 1. Synthesis of PVL-PCL, PVL-PCL-PHOS, PVLPEG-PVLDOXI-PCL-PHOS, and PVLPEG-PVLDOXI-PCL-PHOSFe3O4 Copolymers

(ethylene oxide) (PEG), doxorubicin (DOXI), and magnetic particles all conjugated to the polymeric backbone. Among the three functionalities, PEG and DOXI are grafted as pendant motifs to the lactone copolymer, where as the phosphonoic acid (PHOS) functionality, which is used for anchoring magnetic particle, is functionalized as end group to the copolymer. We believe that the newly designed biodegradable copolymer (PVLPEG-PVLDOXI-PCL-PHOS-Fe 3 O 4 ) having poly(ethylene oxide) (PEG), doxorubicin (DOXI), and a magnetic

nature is expected to behave as smart nanocarrier for both magnetic resonance imaging (MRI) as well as efficient delivery vehicle for cancer therapy. The main objective of this study is to develop an effective method to conjugate magnetic nanoparticle, doxorubicin and poly(ethylene oxide) (PEG) motifs to the biodegradable polyester backbone, which has not been explored. Toward this goal, lactone 1 was synthesized from δ-valerolactone13,9c in the presence of lithiumdiisopropylamide and propargyl bro2704

dx.doi.org/10.1021/ma500384m | Macromolecules 2014, 47, 2703−2711

Macromolecules

Article

the lactone 1, the copolymerization condition with14 εcaprolactone was explored (Scheme 1). The copolymerization of 1 and ε-caprolactone (CL) was carried out in an inert nitrogen atmosphere at room temperature for 48 h by ringopening polymerization method which was initiated by ethanol and catalyzed by Sn(OTf)2 in toluene. Three different types of copolymers namely PVL-PCL1, PVL-PCL2, and PVL-PCL3 were synthesized by changing 1 and CL feed ratios, at constant M/I = 100 and 2 mol % catalyst relative to the initiator. All the copolymerizations were quantitative with more than 90% conversion (Table 1). Among the three copolymers, PVLPCL3 copolymer ([lactone 1]:[ε-CL]: [Sn(OTf)2]:[EtOH] = 50:50:0.5:1) was further used for all the studies. The formation of PVL-PCL3 copolymer was confirmed by GPC and 1H NMR analysis (Figure 1 and S10 (Supporting Information)). The GPC trace of PVL-PCL3 copolymer was observed as unimodal with Mn = 11500 g/mol and PDI = 1.1 using polystyrene standards (Figure 1a). The observed narrow PDI indicated the

Table 1. GPC Data of all Copolymers run

feed ratio (1:CLa)

Mnb (targeted)

Mnc (observed)

PDId

PVL-PCL1 PVL-PCL2 PVL-PCL3

10:90 75:25 50:50

11640 13200 12600

9800 12500 11500

1.25 1.38 1.11

a ε-caprolactone. bTheoretical number-average molecular weight (Mn). cMn was determined by GPC in THF relative to linear poly(methyl methacrylate) standards. dPolydisperisty index (PDI) was determined by GPC in THF relative to linear poly(methyl methacrylate) standards.

mide at −78 °C. The crude product was purified first by column chromatography, followed by distillation under reduced pressure at 160 °C, produced lactone (1) as a colorless, viscous liquid with 45% yield. The formation of 1 was confirmed by observing a new signal at δ 2.0 ppm in 1H NMR spectroscopy corresponding to the terminal acetylene protons. After isolating

Figure 1. (a) Representative gel permeation chromatogram of PVL-PCL3; (b) 1H NMR spectrum of PVLPEG-PVLDOXI-PCL-PHOS copolymer in CD3OD. 2705

dx.doi.org/10.1021/ma500384m | Macromolecules 2014, 47, 2703−2711

Macromolecules

Article

Figure 2. Comparative 31P NMR spectra of PHO, PHOS, and PVL-PCL-PHOS.

butyl 2-(4-azidobenzoyl) hydrazine carboxylate was prepared by coupling reaction between 4-azidobenzoic acid and bocprotected hydrazine in the presence of catalysts N-(3(dimethylamino)propyl)-N′-ethylcarbodiimide hydrochloride/ 1-hydroxybenzotriazole. In 1H NMR spectroscopy (Figure S3), disappearance of signal at δ 13 ppm and appearance of a new signal at δ 1.4 ppm (boc-methyl protons) suggested the formation of tertiary butyl 2-(4-azidobenzoyl) hydrazine carboxylate. It was further confirmed by FTIR and 13C NMR spectroscopy (Figure S4). The boc-deprotection of tert-butyl 2(4-azidobenzoyl) hydrazine carboxylate was carried out by treating it with trifluoroacetic acid in dichloromethane as solvent. The absence of signal at δ 1.4 ppm for boc-methyl proton and appearance of new signal due to -NH-NH2 proton at δ 3.9 ppm in 1H NMR spectrum of (Figure S5), which suggested the complete deprotection of tertiary butyl group from compound 3. It was further confirmed by FTIR and 13C NMR spectroscopy (Figure S6). Compound 4 with acylhydrazine linker was prepared by addition of doxorubicin hydrochloride and 4-azidobenzodrazide in methanol in the presence of catalytic amount of trifluoroacetic acid. The product was confirmed by 1H NMR and FTIR spectroscopy. The signals at δ 7.94−7.95 (m, 2H), 7.79−7.80 (m, 2H), 7.77− 7.78 (m, 1H), and 7.36−7.39 (m, 1H) ppm were responsible for DOXI aromatic group protons which indicated the formation of product (Figure S7). Also, in FTIR spectroscopy, the stretching frequency at 2090 cm−1 (due to azide), that indicated the formation of compound 4 (DOXI-N3) with acylhydrazine linker. Finally, compound 6 (Scheme S2) was prepared by coupling reaction of PEG-550 monomethyl ether and 4-azidobenzodrazide in the presence of DCC)/DMAP in DCM solvent. It was characterized by 1H NMR (Figure S8), FTIR and 13C NMR spectroscopy. From the FTIR spectrum of compound 6, the stretching frequency of azide was observed at 2095 cm−1, which confirmed the product formation. It was further confirmed by 13C NMR spectroscopy (Figure S9). A signal at δ 50.9 ppm corresponded to methylene protons, which indicated the formation of the compound 6. After successful synthesis of compounds 4 and 6, experimental condition for the click chemistry between azide (compounds 4 and 6) and acetylene (PVL-PCL-PHOS) was

controlled polymerization of the monomers to produce PVLPCL3. The molar ratio of 1 in PVL-PCL3 was found by measuring the integral value of acetylene proton at 2.0 ppm and comparing it with the integral value of CH2−O protons at δ 4.03 ppm in 1H NMR spectroscopy (Figure S10). To incorporate PHOS functionality (Scheme 1) in PVLPCL3, 6-phosphonohexanoic acid was prepared by refluxing 6(diethoxyphosphoryl)hexanoic acid ethyl ester with concentrated hydrochloric acid. The formation of product was confirmed by 1 H NMR, 31P NMR, and FTIR spectroscopy. Disappearance of signal at δ 32.7 ppm corresponds to phosphate ester, and formation of a new signal at δ 26.5 ppm corresponds to phosphonic acid in 31P NMR spectroscopy confirmed the product formation (Figure 2). Next, PVL-PCLPHOS was prepared by coupling PVL-PCL3 with carboxylic derivative of phosphonohexanoic acid in the presence of dicyclohexylcarbodiimide (DCC)/4-dimethylaminopyridine (DMAP) in dichloromethane solvent. The PVL-PCL-PHOS copolymer was purified by reprecipitation method using cold hexane as a solvent. The product formation was confirmed by 1 H NMR (Figure S11), FTIR and 31P NMR spectroscopy. The 31 P NMR spectrum of PVL-PCL-PHOS copolymer showed a signal at δ 26.5 ppm which was due to phosphonic acid in compound 5 (Figure 2). It indicated the attachment of phosphonohexanoic acid with PVL-PCL3 copolymer. Also, in FTIR spectroscopy the appearance of the characteristic IR bands at 1261 and 1015 cm−1 were responsible for to PO bond and the P−O bond, respectively (Figure S12c). Both 31P NMR and FTIR spectroscopy confirmed the formation of PVLPCL-PHOS copolymer. Next, the possibilities of attaching DOXI motif15 to the PVLPCL-PHOS random copolymer was explored (Scheme 1). Towards this motivation azide-containing doxorubicin with acylhydrazine linker (DOXI-N3) was synthesized in four steps as shown in the Supporting Information, Scheme S1. 4Azidobenzoic acid was prepared by using well-known diazotization16 reaction of 4-aminobenzoic acid. The % of yield was observed maximum (90%) when NaN3 was added slowly or dropwise fashion for 1 h using additional dropping funnel. The product formation was monitored by 1H NMR, FTIR and 13C NMR spectroscopy (Figure S1 and S2). Tertiary 2706

dx.doi.org/10.1021/ma500384m | Macromolecules 2014, 47, 2703−2711

Macromolecules

Article

PHOS copolymer was characterized by 1H NMR and FTIR spectroscopy. In 1 H NMR spectroscopy of PVLPEGPVLDOXI-PCL-PHOS copolymer revealed all characteristic peaks of DOXI, PEG, phosphate, and the aliphatic polyester backbone (Figure 1b). In addition, new signal at δ 7.37 ppm observed due to the triazole proton which confirmed the formation of PVLPEG-PVLDOXI-PCL-PHOS copolymer. Also, in the FTIR spectroscopy (Figure S12a), the disappearance of azide signal at 2095 cm−1 in PVLPEG-PVLDOXIPCL-PHOS copolymer confirmed the successful click reaction. It was further confirmed by GPC analysis of PVLPEGPVLDOXI-PCL-PHOS copolymer using polystyrene as a standards, which indicated a substantial increase in molecular weight, from Mn = 11 500 g/mol (before conjugation) to Mn = 20 400 g/mol (after conjugation), with relatively narrow polydispersity (PDI) = 1.9 (Figure S16). The self-aggregation study of PVLPEG-PVLDOXI−PCL− PHOS copolymer was carried out in water. One mg of PVLPEG-PVLDOXI−PCL−PHOS copolymer was dissolved in 1 mL of water. Then the solution was stirred until a clear solution was obtained. The particle size was measured around 70 nm with 0.34 PDI using dynamic light scattering (DLS). Further the morphology of the aggregates was determined by SEM (Figure 3a and b) as well as TEM (Figure 3c, d and e). From TEM and SEM analysis, the shape of nano aggregates were observed as nanocapsules and the size of these nanocapsules was around 70 nm, which was in good agreement with DLS measurement (Figure S15a). During the magnification of the image under the TEM, the capsules started degrading due to beam daming under the electron beam (Figure 3e). A recent report suggested that worm, or rod, shaped aggregates showed a high loading capacity of hydrophobic objects per micelle as well as long in vivo circulation capabilities.19 So the observed nanocapsules would be very interesting to explore it further as these type of morphologies would also exhibit in vivo circulation properties due to their unique nanocapsule shape. To test the stimuli responsive nature of newly designed nanocarrier, in vitro drug release studies were carried out before attaching the magnetic particles to PVLPEG-PVLDOXI-PCLPHOS copolymer. For the drug release profile of PVLPEGPVLDOXI-PCL-PHOS copolymer was monitored at pH 7.4, as well as acidic condition. 1 mg sample of PVLPEGPVLDOXI-PCL-PHOS copolymer was dissolved in 1 mL of distilled water. This solution was loaded in dialysis tube (3,500; Dalton cutoff) and dialyzed against 100 mL of a buffer solution whose pH was maintained to 6.0. From the above solution, 2.5 mL of solution was taken and its absorption spectrum was obtained from UV spectroscopy. The absorbance was observed at 480 nm, as an indication of the release of doxorubicin. Fluorescence also recorded by exciting the solution at 510 nm. Emissions of the free DOXI released from PVLPEGPVLDOXI-PCL-PHOS were observed at 560 and 590 nm. The sample was then poured back to maintain the volume of the solution constant. This procedure was repeated for every 1 h time interval up to 12 h. After 12 h it was observed that there was no significance increase in the intensity of fluorescence. Similar procedure was carried out for the drug release at pH 7.4. All the results are shown in Figure S14. The DOXI release from PVLPEG-PVLDOXI-PCL-PHOS copolymer at pH 7.4 was observed less than 10%, it was interesting to note this observation as it clearly demonstrated the PVLPEGPVLDOXI-PCL-PHOS system’s stability in the physiological

Figure 3. (a and b) SEM images of PVLPEG-PVLDOXI-PCL-PHOS copolymer; (c−e) TEM images of PVLPEG-PVLDOXI-PCL-PHOS; (f) Proposed cartoon representation for the observed nanocapsules.

explored. In general, most of the azide and acetylene coupling reaction were performed either in water or mixed polar solvents.9c For the first time, in our experiments, the click reaction was performed in tetrahydrofuran and water (1:1) solvent sytems. Click reaction was carried out by targeting 50 mol % DOXI and 50 mol % PEG, in the presence of sodium ascorbate and copper(II) sulfate pentahydrate. The reaction mixture was allowed to stirr at room temperature for 24 h. The unreacted compounds 4 and 6 were removed by dialysis in phosphate buffer solution. Water was evaporated using high vacuum pump to get the pure and dry PVLPEG-PVLDOXIPCL-PHOS copolymer. The PVLPEG-PVLDOXI-PCL2707

dx.doi.org/10.1021/ma500384m | Macromolecules 2014, 47, 2703−2711

Macromolecules

Article

Figure 4. (a) TGA data of PVLPEG-PVLDOXI-PCL-PHOS copolymer (A), PVLPEG-PVLDOXI-PCL-PHOS-Fe3O4 copolymer (B), PVLPEGPVLDOXI-PCL-PHOS copolymer + Fe3O4 physical mixture (C) and Fe3O4 nanoparticles alone (D); (b) SQUID data of PVLPEG-PVLDOXIPCL-PHOS-Fe3O4; insert-Demonstration of influence of permanent magnet on PVLPEG-PVLDOXI-PCL-PHOS-Fe3O4 solution; (c) EDX of PVLPEG-PVLDOXI-PCL-PHOS-Fe3O4 in water; insert-SEM of PVLPEG-PVLDOXI−PCL−PHOS-Fe3O4; (d) SEM images of PVLPEGPVLDOXI-PCL-PHOS-Fe3O4 in water (scale bar =200 nm); (e−g) TEM images of PVLPEG-PVLDOXI-PCL−-PHOS-Fe3O4 in water (scale bar =100 nm).

stability up to 190 °C, which was 40 °C higher than the PVLPEG-PVLDOXI-PCL-PHOS copolymer. We believed that the observed increase in stability was due to the conjugation of particles to the −OH motifs of phosphonic acid in PVLPEG-PVLDOXI−PCL−PHOS copolymer. The −OH motifs of the copolymer got degraded at 150 °C, whereas after attachment of Fe3O4 nanoparticles, thermal stability of Fe3O4 conjugated copolymer increased up to 190 °C. Our proposal was further confirmed by a control experiment. TGA of physical mixture of Fe3O4 nanoparticles and PVLPEGPVLDOXI−PCL−PHOS was performed (Figure 4a (C)). It was interesting to note that the initial degradation point of the physical mixture was observed at 150 °C. Since it was a physical mixture, the degradation pattern was similar to the individual profiles. From wt % of the final residue, the amount of Fe3O4 conjugated to the polymer was calculated. The observed TGA results were further confirmed by FTIR spectroscopy (Figure S13). The appearance of the characteristic bands of Fe3O4 at 640 cm−1 in FTIR spectrum from PVLPEG-PVLDOXI-PCLPHOS-Fe3O4 copolymer confirmed the conjugation of Fe3O4 nanoparticles to the PVLPEG-PVLDOXI-PCL-PHOS copolymer. The conjugation of Fe3O4 nanoparticles to the PVLPEGPVLDOXI-PCL-PHOS copolymer was further confirmed by SEM, EDX, DLS and TEM. From SEM experiments (Figure 4d), it was observed that the nonocarrier size increases from 70 nm (before conjugation) to 110 nm (after conjugation). This

condition of human body. It was also very interesting to note that the maximum 60% drug was release at pH 6.0 as compared to pH 7.4, suggested the importance of having the acid-labile acylhydrazine linker in the PVLPEG-PVLDOXI-PCL-PHOS copolymer. After demonstrating the stimuli responsive nature of PVLPEG-PVLDOXI-PCL-PHOS copolymer, Fe3O4 nanoparticle conjugation was carried out following a known procedure17,18 (Scheme 1). Magnetic nanoparticles were purchased from Sigma-Aldrich which was having size less than 40 nm. Fe3O4 nanoparticles were functionalized with the PVLPEG-PVLDOXI-PCL-PHOS copolymer. Thermogravimetric analysis (TGA) on PVLPEG-PVLDOXI-PCL-PHOSFe3O4 confirmed the conjugation of magnetic nanoparticle to the PVLPEG-PVLDOXI-PCL-PHOS copolymer (Figure 4a(B)). To prove that, TGA analysis of Fe3O4 nanoparticles alone was performed. It was observed that there was no thermal degradation up to 500 °C (Figure 4a(D)). Similarly, TGA of PVLPEG-PVLDOXI-PCL-PHOS copolymer alone was performed. The copolymer was stable up to 150 °C, after that the first degradation point was started (Figure 4a(A)). The weight loss of the polymer was observed near about 65% when it reached the temperature 280 °C. And then the second degradation point started at 340 °C. The remaining 25% of the polymer degraded between the temperatures 340 to 400 °C. The TGA analysis of Fe3O4 conjugated copolymer showed 2708

dx.doi.org/10.1021/ma500384m | Macromolecules 2014, 47, 2703−2711

Macromolecules

Article

Figure 5. (a) Cartoon representation of the magnetic field induced nanocarrier internalization; (b) CLSM images of control molecule. The nucleus is stained with DAPI; (c) uptake of PVLPEG-PVLDOXI-PCL-PHOS copolymer in 4T cells with different time intervals (12, 24, 36, and 48 h); (d) cytotoxicity profile of PVLPEG-PVLDOXI-PCL-PHOS copolymer in 4T cells.

expected to produce gradient magnetic field. It was very interesting to observe the movement of nanocarriers in aqueous solution when they were introduced to the permanent magnet (insert Figure 4b). We expected that newly designed PVLPEGPVLDOXI−PCL−PHOS-Fe3O4 nanocarrier would be effectively utilized for enhanced tumor penetration (Figure 5a). Nanocapsule from PVLPEG-PVLDOXI−PCL−PHOS with pH linker provided the additional edge over the free drug as it could be released slowly inside the cancerous cell. Next, to

was well supported by DLS (Figure S15b) and TEM (Figure 4e−g). The characteristic energy dispersive X-ray (EDX) spectrum of PVLPEG-PVLDOXI-PCL-PHOS-Fe3O4 confirmed the presence of Fe (Figure 4c). The magnetization study of PVLPEG-PVLDOXI-PCLPHOS-Fe3O4 nanocarriers was measured by magnetic property measurement system (MPMS) magnetometry at 300 K (Figure 4b). From magnetization (M), it was confirmed that the nanocarrier showed superparamagnetic nature, and hence it was 2709

dx.doi.org/10.1021/ma500384m | Macromolecules 2014, 47, 2703−2711

Macromolecules

Article

prove the magnetic field induced drug delivery, PVLPEGPVLDOXI-PCL-PHOS-Fe3O4 incubated 4T cells were analyzed using flow cytometer (Supporting Information, Figures S17 and S18). It was studied in cell lines of a mouse mammary gland cancer cell line (4T). 4T cells in suspension with nanocapsule were taken in MACS magnetic column (Miltenyi Biotec) and kept in the presence of magnetic field as shown in (Figure 5a). 4T cells in suspension with nanocapsule without magnetic field was treated as control. Since the nanocapsule could give emission in the red spectrum due to DOX motif, APC (Allophycocyanin; Ex- 640 nm and Em-660 nm) channel was used to perform the flow cytometry analysis. Propidium iodide experiment confirmed that only living cell populations were being studied for the drug internalization under the magnetic field (Supporting Information, Figure S17). It was very interesting to note the clear intensity shift over cells while comparing “with magnet” and “without magnet” in APC channel. On the basis of the intensity shift, it was very surprising to observe that the much greater internalization of the nanocapsule in the presence of magnetic field (Supporting Information, Figure S18). It is well-known that the internalization concentration of a nanocapsule on a target cell actually depends on the concentration of nanocapsules in extracellular circumstance.20 So we hypothesized that due to the magnetic field guiding transfer efficiency of drug vectors was improved. The cellular uptake behavior of biodegradable nanocapsule was analyzed using confocal laser scanning microscope (Zeiss, LSM 710). It was obvious from the CLSM images (Figure 5b and c) that the amount of internalization of DOXI on 4T cells increased with increasing time as well as magnetic field in comparison with the control molecule. The cell growth effect PVLPEG-PVLDOXI-PCL-PHOS copolymer was determined using trypan blue exclusion method. The effect of cell viability (Figure 5d) was tested in 4T cells by incubating the nanocapsules from the copolymer with different concentration, (250, 500, and 750 μg). Most interestingly, the DOXI conjugated nanocapsules showed an enhanced antitumor activity, which could be due to a prolonged retention of the nanocarrier in the nucleus.

study. This material is available free of charge via the Internet at http://pubs.acs.org.

■

*(R.S.) E-mail: [email protected]. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS G.M.N. thanks UGC for the research fellowship and N.V.K. thank CSIR for the research fellowship. H.D. thanks the DST. R.S. thanks the Department of Science and Technology, New Delhi, India, for a Ramanujan fellowship. R.S. and J.D.S. thank the IISER-Kolkata for providing the infrastructure and start up funding. All authors thank Dr. Chiranjib Mitra, Department of Physical Science, IISER-Kolkata, for the MPMS magnetometry measurements and Koushik Chatterjee and Tanmoy Dalui for the confocal studies.

■

REFERENCES

(1) (a) Duncan, R. Nat. Rev. Cancer 2006, 6, 688. (b) Duncan, R. Nat. Rev. Drug Discovery 2003, 2, 347. (c) Duncan, R.; Kopecek. J. Nat. Rev. 1984, 57, 51. (2) (a) Duncan, R. Anti-Cancer Drugs 1992, 3, 175. (b) Nowicka, A. M.; Kowalczyk, A.; Jarzebinska, A.; Donten, M.; Krysinski, P.; Stojek, Z.; Augustin, E.; Mazerska, Z. Biomacromolecules 2013, 14, 828. (c) Wei, H.; Zhuo, R. X.; Zhang, X. Z. Prog. Polym. Sci. 2013, 38, 503. (d) Ge, Z.; Liu, S. Chem. Soc. Rev. 2013, 42, 7289. (3) (a) Singla, A. K.; Garg, A.; Aggarwal, D. Int. J. Pharm. 2002, 235, 179. (b) Stukel, J. M.; Li, R. C.; Maynard, H. D.; Caplan, M. R. Biomacromolecules 2010, 11, 160. (c) Li, D.; Sun, H.; Ding, J.; Tang, Z.; Zhang, Y.; Xu, W.; Zhuang, X.; Chen, X. Acta Biomater. 2013, 9, 8875. (4) (a) Gillies, E. R.; Frechet, J. M. J. Bioconjugate Chem. 2005, 16, 361. (b) Gillies, E. R.; Frechet, J. M. J. Chem. Commun. 2003, 14, 1640. (c) Lehn, J.-M.; Eliseev, A. Science 2001, 291, 2331. (d) Gillies, E. R.; Frechet, J. M. J. Pure Appl. Chem. 2004, 76, 1295. (5) (a) Maeda, H.; Wu, J.; Sawa, T.; Matsumura, Y.; Hori, K. J. Controlled Release 2000, 65, 271. (b) Satav, S. S.; Bhat, S.; Thayumanavan, S. Biomacromolecules 2010, 11, 1735. (6) Maisie, J.; Samantha, M.; Todd, E. Chem. Commun. 2010, 46, 1377. (7) (a) Grubbs, R. H. Handbook of Metathesis; Wiley-VCH: NewYork, 2003; Vol. 3. (b) Watson, K. J.; Park, S.-J.; Im, J.-H.; Nguyen, S. T. Macromolecules 2001, 34, 3507. (c) Mortell, K. H.; Gingras, M.; Kiessling, L. L. J. Am. Chem. Soc. 1994, 116, 12053. (8) Gillies, E. R.; Goodwin, A. P.; Frechet, J. M. J. Bioconjugate Chem. 2004, 15, 1254. (9) (a) Albertsson, A. C.; Varma, I. K. Biomacromolecules 2003, 4, 1466. (b) Greenwald, D.; Shumway, S.; Albear, P.; Gottlieb, L. J. Surg. Res. 1994, 56, 372. (c) Parrish, B.; Breitenkamp, R. B.; Emrick, T. J. Am. Chem. Soc. 2005, 127, 7404. (10) (a) Padilla De Jesus, O. L.; Ihre, H. R.; Gagne, L.; Frechet, J. M. J.; Szoka, F. C. Bioconjugate Chem. 2002, 13, 453. (b) Uhrich, K. E.; Cannizzaro, S. M.; Langer, R. S.; Shakesheff, K. M. Chem. Rev. 1999, 99, 3181. (11) (a) Mizutani, M.; Arnold, S. C.; Matsuda, T. Biomacromolecules 2002, 3, 668. (b) Nederberg, F.; Bowden, T.; Hilborn, J. Macromolecules 2004, 37, 954. (c) Cordova, A.; Iversen, T.; Hult, K. Polymer 1999, 40, 6709. (12) (a) Rao, V. N.; Kishore, A.; Sarkar, S.; Das Sarma, J.; Shunmugam, R. Biomacromolecules 2012, 13, 2933. (b) Mane, S. R.; Rao, V. N.; Chatterjee, K.; Dinda, H.; Nag, S.; Kishore, A.; Das Sarma, J.; Shunmugam, R. J. Mater. Chem. 2012, 22, 19639. (13) Herrmann, J. L.; Schlessinger, R. H. Chem. Commun. 1973, 711.

■

CONCLUSION In conclusion, we have designed and successfully synthesized a PVLPEG-PVLDOXI-PCL-PHOS-Fe3O4 copolymer which can be used as a nanocapsule for intracellular sustained delivery of the antitumor drug DOXI. Most importantly, both Fe3O4, and DOXI, are efficiently conjugated to the biodegradable polymeric backbone. The resulting DOXI conjugated nanocapsule exhibits controlled and pH-responsive drug-release profile. The results clearly show the importance of having the acylhydrazine linker which helps to release the drug at the mild acidic condition. From the cell viability studies, it is clear that the nanocapsules are very effective in inhibiting tumor cell growth at low concentration in vitro. The newly designed biodegradable nanocapsules provide a powerful magnetic moment under moderate gradient magnetic fields. We envision that our unique approach may open up a new avenue for more effective cancer therapy through well-informed decisionmaking.

■

AUTHOR INFORMATION

Corresponding Author

ASSOCIATED CONTENT

S Supporting Information *

Experimental section, synthesis scheme of all monomers, 1H NMR, 13C NMR and IR spectra of monomers, and a dialysis 2710

dx.doi.org/10.1021/ma500384m | Macromolecules 2014, 47, 2703−2711

Macromolecules

Article

(14) Darcos, V.; El Habnouni, S.; Nottelet, B.; El Ghzaoui, A.; Coudane, J. Polym. Chem. 2010, 1, 280. (15) Parrish, B.; Emrick, T. Bioconjugate Chem. 2006, 18, 263. (16) Li, L.; Han, J.; Nguyen, B.; Burgess, K. J. Org. Chem. 2008, 73, 1963. (17) Peng, R.; Zhang, W.; Ran, Q.; Liang, C.; Jing, L.; Ye, S.; Xian, Y. Langmuir 2011, 27, 2910. (18) (a) Yoo, D.; Lee, J. H.; Shin, T. H.; Cheon, J. Acc. Chem. Res. 2011, 44, 863. (b) Ho, D.; Sun, X.; Sun, S. Acc. Chem. Res. 2011, 44, 875. (c) Bilalis, P.; Chatzipavlidis, A.; Tziveleka, L. A.; Boukosa, N.; Kordas, G. J. Mater. Chem. 2012, 22, 13451. (d) Ma, W. F.; Wu, K. Y.; Tang, J.; Li, D.; Wei, C.; Guo, J.; Wang, S. L.; Wang, C. C. J. Mater. Chem. 2012, 22, 15206. (19) Geng, Y.; Dalhaimer, P.; Cai, S.; Tsai, R.; Tewari, M.; Minko, T.; Discher, D. E. Nat. Nanotechnol. 2007, 2, 249. (20) Li, R.; Wu, R.; Zhao, L.; Hu, Z.; Guo, S.; Pan, X.; Zou, H. Carbon 2011, 49, 1797.

2711

dx.doi.org/10.1021/ma500384m | Macromolecules 2014, 47, 2703−2711