Triple Block Nanocarrier Platform for Synergistic Cancer Therapy of

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Triple Block Nanocarrier Platform for Synergistic Cancer Therapy of Antagonistic Drugs Bapurao Surnar, and Manickam Jayakannan Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.6b01608 • Publication Date (Web): 11 Nov 2016 Downloaded from http://pubs.acs.org on November 12, 2016

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Triple Block Nanocarrier Platform for Synergistic Cancer Therapy of Antagonistic Drugs Bapurao Surnar and Manickam Jayakannan*1

Department of Chemistry Indian Institute of Science Education and Research (IISER) Pune Dr. Homi Bhabha Road Pune 411008, Maharashtra, INDIA

Abstract A unique biodegradable triple block nanocarrier (TBN) is designed and developed for synergistic combination therapy of antagonistic drugs for cancer treatment. The TBN was built with hydrophilic polyethyleneglycol (PEG) outer shell; a middle hydrophobic and biodegradable polycaprolactone (PCL) block for encapsulating anthracycline anticancer drug like doxorubicin (DOX), and an inner carboxylicfunctionalized polycaprolactone (CPCL) core for cisplatin (CP) drug conjugation. TBN-cisplatin drug conjugate self-assembled as stable nanoparticles in saline (also in PBS) wherein the hydrophobic PCL block functions as a shield for Pt-drug stability against GSH detoxification. Enzymatic-biodegradation of TBN exclusively occurred at the intracellular environment to deliver both cisplatin (CP) and doxorubicin (DOX) simultaneously to the nucleus. As a result, the TBN-cisplatin conjugate and its DOX loaded nanoparticles accomplished 100 % cell growth inhibition in GSH overexpressed breast cancer cells. Combination therapy revealed that free drugs were antagonistic to each other whereas the dual drug loaded TBN exhibited excellent synergistic cell killing at much lower drug concentrations in breast cancer cells. Confocal microscopic analysis confirmed the localization of drugs in the cytoplasm and at peri-nuclear site. Flow cytometry analysis revealed that the drugs were taken up four folds better while delivering them from TBN platform compared to free form. The TBNs approach is a perfect platform to overcome the GSH detoxification in Ptdrugs and enable the co-delivery of antagonistic drugs like cisplatin and DOX from single polymer dose to accomplish synergistic killing in breast cancer cells. 1

Corresponding author: [email protected]

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Keywords: Platinum drug delivery, block copolymers, enzyme-responsiveness, combination therapy, synergistic effect. Introduction Cisplatin administration has major obstacle in breast and ovarian cancer patients because of the cancer tissue resistance to Pt-drugs at the second and third levels of chemotherapy treatment.1 This obstacle directly influences on the survival of the ovarian patients and more than 45 % of the patients are vulnerable to metastasis within a short span of 5-10 years.2 Nonspecific and poor transportation of Pt-drug; detoxification of cisplatin drug by glutathione (GSH) residues, and DNA-repair mechanism were identified as some of the major issues associated with drug adminsitration.3 To address these problems, polymer molecular nano-carriers

17-23

4-16

and small

were developed to increase the cellular uptake of Pt-drugs via

enhanced permeability and retention (EPR) effect.24-26 Pt-drugs were also combined with doxorubicin (DOX) and paclitaxel to improve their therapeutic efficacies.27-28Carboxylic substituted polysaccharide nanoparticles29 and chitosan-polymethacrylic acid blends30 were developed for co-delivery of DOX and cisplatin. However, the co-delivery of pharmacokinetically different drugs like cisplatin (metal drug) and polyaromatic drugs was found to be a difficult task due to their antagonistic nature and also variation in solubility parameters.5 Additionally, the breast cancer cells (also ovarian cancer cells) are over expressed with GSH (20 mM) compared to normal cells (0.5 to 1.0 mM);31-32 thus, the Ptdrug is readily detoxified by GSH and excreted from the cytoplasm prior to their reaching at the nucleus.33 Further, most of the Pt-drug delivery nano-carriers were designed from nonbiodegradable polymer backbone; thus, their drug releasing mechanism at the intracellular level was not clearly understood. To address these important problems, it is urgently required to design new polymer nano-carrier to enable the combination therapy of cisplatin and DNAintercalating drug DOX together. This would facilitate the delivery of drugs against the antagonistic effect, stabilise the drug against GSH detoxification, and deliver them by enzyme at the intracellular compartments.To accomplish these goals, here we propose a new triple-block nano-carrier (TBN) design with following distinct features: (i) PEG hydrophilic shell at the periphery for the aqueous solubility of the nanoparticles for drug administration, (ii) hydrophobic PCL middle block for the encapsulation of DNA intercalating drug such as DOX, and (iii) carboxylic functionalized PCL block (CPCL) for chemical conjugation of cisplatin at the core of the TBN. In this design both PCL and CPCL blocks are aliphatic polyesters; thus, they are enzymatically-biodegradable at the intracellular lysosomal 2 ACS Paragon Plus Environment

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compartments for drug delivery. Further, the middle hydrophobic PCL block also behaves as a shield against the attack of GSH in the cytoplasm and stabilizes the cisplatin drug in the active form for cancer treatment. This multipurpose TBN design for combination drug delivery is shown in figure 1.

Figure 1. Triple block nanocarrier (TBN) design for combination therapy of cisplatin and DOX and accomplishing synergetic cancer therapy from single polymer platform. This design was conceived by us based on our expertise in developing biodegradable polycaprolactone block copolymer nano-architectures for cancer therapy.34-39 In the present investigation, novel PEG-b-PCL-b-CPCL triblock design was conceptualized and synthesized through controlled ring-opening polymerization process in good molecular weights with narrow polydispersity. This design allowed us to simultaneously conjugate cisplatin drug and also physically load DOX in a single polymer dose. The rationale of TBN design was investigated in detail with appropriate structural engineering and the right structure was optimized for successful dual loading of drugs. In vitro studies in cancerous cell lines revealed that the free drugs are antagonistic to each other whereas their triple-layer block copolymer nanoparticles exhibited excellent synergistic killing in breast cancer cells. Cellular uptake by confocal microscope and flow cytometry analysis confirmed the internalization of the triple block nanoparticles and the accumulation of drugs at the peri-nuclear environment. This approach has opened a novel platform of triple-block nano-carrier (TBN) design for the combination therapy of cisplatin and doxorubicin (DOX) in cisplatin-resistant breast cancer. 3 ACS Paragon Plus Environment

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Experimental Section Synthesis of PEG-b-PCL-b-BPCL triblock polymers: The typical synthetic

procedure is elucidated for PEG2000-b-PCL50-b-BPCL100, this triblock synthesized in two steps. In first step, [Mo]/[Io] is kept as 50 i.e. monomer 1 (caprolactone, M1) to initiator PEG which provide diblock with 50 PCL units. To this block monomer 2 (substituted caprolactone, M2) was added considering the formed diblock as a macroinitiator [Mo]/[Io] is kept as 100. The initiator PEG2000 (70.16 mg, 0.035 mmol), catalyst Sn(Oct)2 (7.0 mg, 0.0175 mmol) and monomer 1 (200 mg, 1.75 mmol) were taken in a flame-dried Schlenk tube and carried out the polymerization as reported earlier.36 Substituted caprolactone monomer 2 (900 mg, 3.50 mmol) was added to the schlenk tube under high inert conditions and temperature of oil bath was raised to 130 o

C. Reaction was continued for additional 24 h to achieve triblock copolymer. The

polymer was cooled, and purified by dissolving in THF and precipitated in cold MeOH. Yield = 800 mg (72 %). 1H-NMR (400 MHz, CDCl3) δ ppm: 4.13 (m, 2 H), 4.04 (m, 1 H) 3.63 (m, 3.68 H), 3.43 (m, 1 H), 3.36 (s, 3 H), 2.44 (t, 2 H), 2.36 (t, 2 H), 2.29 (m, 1 H), 1.77 (m, 4 H), 1.67 (m, 2 H), 1.44 (s, 9 H, t-butyl), 1.36 (m, 1.5 H). 13

C-NMR (100 MHz, CDCl3): 173.5, 173.4, 170.8, 80.5, 75.5, 70.6, 64.8, 64.1, 61.2,

36.5, 34.1, 32.9, 29.7, 28.2, 25.8, 24.4. FT-IR (cm−1): 2978, 2862, 1790, 1704 (C=O ester), 1423, 1318, 1146, 1156, 1100, 949, 900, 845, and 721. GPC molecular weights: Mn = 15,000 g/mol, Mw = 27,100 g/mol and Mw/Mn= 1.80. Other block copolymers were synthesized as described above and these details are given in supporting information. Synthesis

of

PEG-b-PCL-b-CPCL

triblock

polymers:

The

carboxylic

functionalized polymer was prepared by de-protecting the butyl ester side chain units using trifluoroacetic acid in dry DCM as described earlier.36 Yield = 350 mg (88 %). 1

H-NMR (400 MHz, CDCl3) δ: 4.15 (m, 2 H), 4.03 (m, 1 H) 3.70-3.63 (m, 3.65 H),

3.43 (m, 1 H), 3.36 (s, 3 H), 2.58 (t, 2 H), 2.36 (t, 2 H), 2.29 (m, 1 H), 1.77 (m, 4 H), 1.67 (m, 2 H), 1.33 (m, 1.4 H). FT-IR (cm−1): 3447, 2975, 2861, 1795, 1711 (C=O ester and acid), 1423, 1312, 1144, 1156, 1140, 943, 902, 845, and 720. GPC molecular weights: Mn = 12,100 g/mol, Mw = 22,700 g/mol and Mw/Mn= 1.87. Other block copolymers were also de-protected as described above and the details are given in supporting information.


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Synthesis of the Cisplatin Conjugated Triple Block Nanoparticle (TBN-CP): The

cisplatin-triblock copolymer conjugates were prepared by using the sodium salt of the triblock copolymers with aquated cisplatin as described earlier.36 The triple block nanoparticle PEG-b-PCL50-b-CPCL-CP was prepared by solubilising PEG-b-PCL50-bCPCL triblock copolymer (20.0 mg) in 18 mL of DI water and 2 mL of NaOH (1 mg.mL–1) along with constant stirring at 37 °C for 30 min. This was followed by addition of lyophilized aquated cisplatin (7.92 mg, 0.03 mmol) and kept the reaction for additional 24 h at 37 °C. The solution was dialyzed in a 1 kD dialysis bag against Millipore water for 2 days with the water being replaced at regular intervals. The dialyzed solution was filtered through a 0.45 µm filter, lyophilised and stored at 4 oC. Yield = 16 mg (80 %). FT-IR (cm-1): 3350, 2920, 2880, 1669, 1566, 1393, 1360, 1091, 1050, 930, 830 and 544. The other nanoparticles were synthesized by using similar protocal and their details are provided in the supporting information. Preparation of DOX-encapsulated TBNs: The detailed procedure is given for DOX

(DOX.HCl converted to DOX by reacting with triethylamine) encapsulation of PEGb-PCL50-b-CPCL-CP (named as TBN-DOX). For this purpose, 5 mg of lyophilized cisplatin-triblock copolymer conjugate and 0.5 mg of DOX were dissolved in DMSO (1 mL). To this solution, distilled water (4 mL) was added and stirred at 25 °C for 2 h. It was transferred to a dialysis bag (MWCO = 1000) and dialyzed against distilled water for 48 h with replenishing the reservoir with fresh water at regular interval. This procedure facilitated the removal of unencapsulated DOX and the solution in the dialysis bag was filtered, lyophilised and stored at 4 oC. Using similar protocol DOX was loaded into the drug conjugate and triblock polymers (TBN-CP) to produce TBNCP-DOX dual loaded nanoparticles and the detailed information is provided in

supporting information. The drug loading efficiency (DLE) and drug loading content (DLC) were determined as reported earlier.36

In Vitro Drug Release Studies: The cisplatin conjugated (TBN-CP), DOX loaded (TBN-DOX) and dual drug loaded nanoparticles (TBN-CP-DOX) were individually dialyzed in PBS and in presence of esterase (10 U) against PBS at 37 °C in a dialysis bag (3.0 mL). Aliquots of 1.0 mL were withdrawn at regular intervals and replaced with the same amount of buffer. The quantity of cisplatin and DOX released were determined using absorption spectroscopy as mentioned in earlier reports.36, 38 For the 5 ACS Paragon Plus Environment

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estimation of cisplatin release O-phenylenediamine (OPD) Assay was used. The amount of Pt in the sample was quantified by monitoring the absorbance of the OPDPt complex at 706 nm. The procedure and calculations for the DLC and DLE were carried out as explained earlier.36

Cell Viability Assay (MTT Assay) and Cellular Uptake: The cell viability assay

was carried out in order to understand the cytotoxicity of the nascent polymers, free drugs cisplatin (CP) and doxorubicin (DOX), TBN-CP, TBN-DOX and dual drug loaded TBN-CP-DOX in Wt MEF and MCF 7 cell lines. In a 96-well plate, 1000 cells were seeded per well and allowed to adhere in 100 µL of DMEM containing fetal bovine serum (FBS) for 16 h. The media was aspirated and different concentrations of drugs and cargo loaded scaffolds were fed to the cells. The assay was performed using the same protocol as described earlier36 and accordingly the cell viability was determined. For the cellular uptake studies 105 cells were seeded in 6-well plates and incubated with media for 16 h at 37 °C. The cells were then treated with free DOX, TBN-DOX and TBN-CP-DOX followed by 4 h incubation at 37 °C. The cell staining and fixing protocol, imaging and analysis was the same as described earlier.36 Flow Cytometry Measurements: DOX and DOX loaded nanoparticles uptake in

breast cancer cells (MCF 7) was assessed using the flow cytometry cell analyzer. MCF 7 cells were seeded in 6-well plate in DMEM media and incubated for 16 h at 37 °C. Cells were treated with desired concentration (1.84 µM) of free DOX and DOX loaded nanoparticles. After 9 h of incubation, the drug containing media was aspirated and the the cells were digested using 500 µL trypsin followed by 1 min incubation. It was subjected to centrifuge at 10,000 rpm for 5 min. The pellet was re-suspended in 1 mL PBS and flow cytometry studies were carried out by employing the BD LSR Fortessa SORP cell analyzer that is equipped with five lasers and can detect 18 colours simultaneously. The 561 nm laser was used for the excitation of DOX and the band pass filter was chosen as 610 ± 10 nm. The fluorescence histograms were recorded from a population of 10, 000 cells. In order to detect apoptosis induced in MCF 7 cells by the free drugs (CP and DOX) and dual drug loaded nanoparticle TBN-CP-DOX, the annexinV/propidium iodide (PI) assay was carried out using flow cytometry. The cells were incubated, 1X annexin V binding buffer was added and the flow cytometry analysis was performed within 1 h of staining. 6 ACS Paragon Plus Environment

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Results and Discussion Synthesis of cisplatin conjugated and DOX loaded TBN Triple block copolymers were synthesized via the ring opening polymerization (ROP) of caprolactone and butyl ester substituted caprolactone monomer in bulk (or melt) as shown in figure 2a. The butyl ester substituted caprolactone monomer was synthesized from 1,4-cyclohexandiol as reported earlier.34 Polyethylene glycol monomethyl ether MW = 2000 g/mol (PEG) was employed as initiator and Sn(Oct)2 was employed as catalyst. The polymerization of caprolactone by PEG-initiator produced PEG-b-PCL macro-initiator and it initiated the ROP of butyl ester substituted caprolactone monomer to yield novel triblock copolymer PEG-b-PCLx-b-BPCLy in one-pot reaction (B-represents butyl ester substitution, see figure 2a). This triblock was de-protected to yield PEG-b-PCLx-b-CPCLy where C-represents carboxylic substitution; x and y indicate the number of repeating units in PCL and CPCL block, respectively. In these triblock copolymers, CPCL block length was fixed (y = 100) and PCL unit was varied as 25, 50 and 100 (x = 25, 50 and 100) by adjusting the [M]/[I] ratio in the feed for the macro-initiator. Earlier studies from our group have put effort to study the role of PEGs length on the properties of di-block copolymers. Five different initiators with various PEG chain lengths (triethylene glycol, and PEGs of molecular weights varying up to PEG-5000) were used as initiator in ROP of di-block polymers (PEGx-b-CPCL).36Thesediblock copolymers produced stable cisplatin nanoparticles in PBS for PEG-2000 containing hydrophilic blocks. Thus, for the present investigation, PEG-2000 was fixed as the hydrophilic block in the triblock nanoparticle assemblies. 1H-NMR spectra of representative triblock PEG-b-PCL50-bBPCL100 are shown in figure 2b and various protons are assigned. The peak intensities of the PEG part (proton-b at 3.63 ppm) and the PCL protons (proton “a” at 4.03 ppm) were compared in the 1H-NMR spectra of PEG-b-PCL50 macro-initiator (see SF 1) to yield the degree of polymerization (n) for PCL block as 50. In figure 2b, the new peaks for the formation of PEG-b-PCL50-b-BPCL100 triblock were clearly visible. The intensities of proton ‘f’ in the BPCL backbone appeared at 4.13 ppm. The subtraction of peak intensities [(b+g)–f] provides the actual number of protons ‘b’ corresponding to PEG part. The comparison of peak integrals of proton-b and proton-f provided the degree of polymerization (n) for the BPCL backbone in the triblock structure (see figure 2a). Based on this analysis, the PCL and BPCL block lengths were estimated by 1

H-NMR spectra for PEG-b-PCLx-b-CPCLy series (x = 25, 50 and 100 and y = 100) 7 ACS Paragon Plus Environment

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and the details are given in table ST1 in the supporting information (see SF 2 for more NMR spectra).

Figure 2. (a) Synthesis of triblock polymers and their cisplatin-conjugated sample. (b) 1HNMR of PEG-b-PCL50-b-BPCL100 block polymer and (c) GPC chromatograms of PEG-bPCL50 macro-initiator and PEG-b-PCL50-b-BPCL100 block copolymers. Gel permeation chromatography (GPC) was employed for the molecular weight determination of the block copolymers. The GPC chromatograms of PEG-b-PCL50 macro-initiator and the PEG-b-PCL50-b-BPCL100 triblock are shown in figure 2c (for other blocks see SF 3). The triblock copolymer showed distinct chromatogram at lower retention time compared to PEG-b-PCL50 macro-initiator suggesting the formation of higher molecular weight tri-blocks. The number average (Mn) and weight average (Mw) molecular weights and polydispersity (PDI) of polymers are given in table ST1. The triblock copolymer (PEG-b-PCLx-b-CPCL100) exhibited the GPC chromatogram as similar to that of t-butyl ester triblock copolymers suggesting that


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the de-protection into carboxylic functionality did not alter the molecular weight (see SF-4). 1H-NMR spectra of the carboxylic triblock polymer PEG-b-PCL50-b-CPCL100 showed the disappearance of t-butyl at 1.43 ppm (see SF 4). The GPC chromatograms of PEG-b-PCLx-b-CPCL100 triblocks were almost similar to its butyl ester tri-block copolymers (see SF4 and ST1). Thus, the triblock copolymers with variable PCL block lengths at middle and fixed carboxylic PCL were successfully synthesized via the solvent free ROP process.

Figure 3. (a) TGA plots of free cisplatin, triblock PEG-b-PCL50-b-CPCL100 and it‘s cisplatin conjugated sample PEG-b-PCL50-b-CPCL100-CP. (b) DLS histogram, FESEM and HR-TEM images of PEG-b-PCL50-b-CPCL100-CP. (c) DLS histogram and FESEM image of DOX loaded PEG-b-PCL50-b-CPCL100. (d) DLS histogram and FESEM image of DOX loaded in cisplatin-conjugated triblock PEG-b-PCL50-b-CPCL100-CP. The photographs of vials showed red-emission with respect to DOX.

Cisplatin conjugation in the triblock copolymer was accomplished by chelating the pre-synthesized cisplatin aquocomplex with sodium salts triblock copolymer (see figure 1). The triblock copolymer PEG-b-PCL50-b-CPCL100 was subjceted to incubation for 24 h in 1mg/mL NaOH at 37 °C. The GPC plots of the polymers before and after exposure to NaOH did not show any difference in the molecular weight revealing that the polymers were very stable under the cisplatin conjugation reaction condition (see SF 5). The 9 ACS Paragon Plus Environment

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resultant cisplatin-polymer conjugate was dialyzed, lyophilized and stored in dark at 4

°C. The cisplatin conjugation was confirmed by FT-IR spectra (see SF 6). The FT-IR spectra of nascent polymer showed stretching frequency of carbonyl peak at 1720 cm–1 which was shifted to 1558 cm–1 upon complexation with cisplatin (see SF 6).40 Further, the appearance of a new peak at 545 cm–1 with respect to Pt-O–C=O confirmed the polymer-cisplatin drug conjugation.Thermogravimetric plot for TBN, free cisplatin and its cisplatin conjugate TBN-CP are shown in figure 3a. Drug conjugation efficiency (DCE) and drug loading content (DLC) were estimated by thermogravimetric analysis by following the empirical formula developed by Xu et al.41Thermogravimetric plot for PEG-b-PCL50-b-CPCL100, free cisplatin and its cisplatin conjugate PEG-b-PCL50-b-CPCL100-CP are shown in figure 3a. The nascent polymer completely degraded at 400 °C whereas free cisplatin showed only 60 % weight loss for the decomposition of amine and chloride ligands. The cisplatin drugpolymer conjugate exhibited decomposition at 330 °C with respect to polymer and chloride and amine ligands. The residual platinum content in the TGA plots (see Figure 3a) in the cisplatin free drug and cisplatin-polymer drug conjugate PEG-bPCL50-b-CPCL100-CP were obtained as 40 % and 28 %, respectively. From this data, the drug conjugation efficiency (DLE) and drug loading content were obtained as 92 % and 34 % for PEG-b-PCL50-b-CPCL100-CP, respectively (for other triblocks, see ST-2). Dynamic light scattering (DLS) histogram showed self-assembled nanoparticles of 150 ± 10 nm size. FESEM image of cisplatin conjugated triblock exhibited nanoparticular morphologies of 150 ± 5 nm size (see figure 3b) (for other polymers, see SF 7). TEM images of the sample (see Figure 3b) clearly showed dark contrast in the middle and distinct layer at the periphery with respect to PEG-PCL part. The DOX encapsulation was carried out in the triblock copolymer PEG-bPCL50-b-CPCL100by dialysis method in which the polymer and doxorubicin (DOX) were taken in DMSO + water mixture in a semi-permeable dialysis bag and dialyzed for more than 24 h against water. The unencapsulated DOX was removed from the reservoir by continuously replacing with fresh water. The DLC for DOX loading was determined by absorption spectroscopy as 3.8 % and the photographs (see Figure 3c) confirmed the presence of drug.The DLCs of other triblocks PEG-b-PCL25-b-CPCL100 and PEG-b-PCL100-bCPCL100 were obtained as 2.9 and 3.6 %, respectively. This suggtests that minimum of 50 PCL units are sufficenet enough in the triblock design to accomplaihs maximum DOX 10 ACS Paragon Plus Environment

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laoding. Further, the homopolymer CPCL100 and PEG-b-CPCL100 diblock were also subjected to DOX loading (see SF-8). The DLC for DOX encapsulation obtained was very low as 0.4 % and 0.6 % for diblock and homopolymer, respectively. The 10-times higher encapsulation efficiency of doxorubicin in triblock copolymer was attributed to the creation of the hydrophobic PCL middle layer in the triblock nanoparticles. Both CPCL and PEG-bCPCL did not possess perfect triblock geometry; thus, they were inferior for DOX loading. This study revealed that the DOX loading was primarily driven by the triple-block assembly whereas both the homopolymer and diblock do not have appropriate geometry for DOX encapsulation. Thus, the triple block nanoparticle is very unique in encapsulating DOX. The DLS histogram and FESEM images of DOX loaded triblock are shown in figure 3c. DOXloaded nanoparticles showed mono-modal histogram with respect to the average particles size of 135 ± 10 nm. The DOX loading in the cisplatinh conjugated sample PEG-bPCL50-b-CPCL100-CP was carried out in a similar way triblock and the data for the dual loaded triple block nanoparticle are shown in figure 3d. The photograph of the vial showed the encapsulation of DOX in the cisplatin conjugated sample as well. The DLC for the DOX loading in the cisplatin conjuaged sample was determined as 3.8 % which is almost identical to that of the triblock copolymer alone. The mole ratio of the CP and DOX drug content in the dual laoded sample was estimated as 1.0 :18.0 for DOX vs CP, respectively. The DLS histogram of of dual drug loaded sample showed mono-modal histogram of 155 ± 10 nm size (see Figure 3d). FESEM image was found as spherical nano-particles of 140 ± 10 nm size (figure 3d). For simplicity the triblock copolymerPEG-b-PCL50-b-CPCL100, its cisplatin conjuagted sample PEG-b-PCL50-b-CPCL100-CP, DOX loaded sample, and DOX plus cisplatin dual drug loaded samples are referred as TBN, TBN-CP, TBNDOX and TBN-CP-DOX, respectively [TBN= triblock nanoparticle and CP= cisplatin]

Stability, GSH Detoxification and Enzyme-responsiveness Cisplatin-polymer conjugated nanoparticles are expected to encounter three challenges during in vitro cell studies: (i) the destabilization of the Pt-polymer conjugate by the ions presents in the medium, (ii) the GSH detoxification of drugs in the cytoplasm, and (iii) nanoparticles to rupture in the presence of lysosomal enzymes to deliver the drugs (see figure 4a). The stability of the TBN-CP was tested in PBS and saline at 37 °C by dialysis method. The cisplatin is attached on polymer backbone through Pt-OOCpolymer bond which is susceptible to cleave by ions such as Cl- in saline or PO42- in 11 ACS Paragon Plus Environment

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PBS.42 The TBN exhibited more than 90 % stability both in saline and PBS at 37 °C (see figure 4b). Control experiments suggested that the variation of PCLx middle layer from 25 to 100 units enhanced the sability of TBN-CP in PBS from 65 % to 90 % (see SF 9). This pointed out that the hydrophobic PCL block shields against the ion penetration into the cisplatin conjugated core and thus, enhances the stability of the pro-drug manifold. To study the stability of the block copolymer nanoparticle in blood plasma conditions; the TBN-CP nanoparticles were incubated in fetal bovine serum (FBS) at 37 °C for 24 h. As can be seen from the plot in SF 10,there was only 10-15 % release of CP in FBS and hence confirming that they were stable in blood plasma conditions.

Figure 4. (a) TBN cellular internalization and its exposure to three different possibilities at the cellular level. (b) Cumulative release of cisplatin from TBN-CP in PBS, saline and in the presence of esterase enzyme in PBS at 37 °C. (c) The extent of the reaction of cisplatin, oxaliplatin and TBN-CP towards GSH monitored at absorbance maxima at 260 nm. (d) Cumulative drug release profiles of dual loaded TBN-CP-DOX without and with 10 U of esterase enzyme in PBS at 37 °C.


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To study the effect of GSH detoxification on cisplatin,model reactions were performed using free cisplatin, oxaliplatin (cisplatin derivative) and TBN-CP conjugate. The formation of S-Pt bond was monitored using UV-Visible spectroscopy (peak at 260 nm) and the data is summarized in figure 4c (also see SF 11). The GSH detoxification was estimated by (At/A∞) x 100 % where At and A∞ are absorbance at incubation time ‘t’ and at complete reaction, respectively. The increased GSH detoxification in free cisplatin and oxaliplatin clearly showed that these small platinum drugs were not stable towards GSH (figure 4c). On the other hand, the TBN-CP was very stable against GSH detoxification and only less than 2 % drug was detoxified (see figure 4c). The excellent stability of TBN-CP is attributed to the PCL hydrophobic shield against the GSH penetration into the cisplatin core. This experiment directly proves that the TBN is an excellent design to preserve the cisplatin drug against GSH detoxification.Esterase enzymes are largely abundant in the lysosomes and they are capable of cleaving aliphatic polyester PCL backbone in TBN to rupture the drug loaded nanoparticles to release the cargoes (see figure 4a).43-45To investigate the cisplatin and DOX release, the drug loaded TBN were subjected to release studies in PBS alone and in the presence of 10 U of esterase enzyme in PBS at 37 °C. The cumulative release profiles revealed that the TBN backbone ruptured in the presence of esterase enzyme and the cisplatin released from TBN-CP in a controlled manner for 48 h (see figure 4b). The TBN-DOX polymer nanoparticles also exhibited similar selectivity for DOX release in the presence of esterase enzyme (see SF 12). The in vitro release experiment was carried out to study the stablity of TBN-DOX at very low pH 4.0 at 37 °C for 24 h. The release kinetics clearly showed that the DOX release was < 25 % at pH = 4.0 (see SF 13) which is almost similar to the release profiles at pH= 7.4 in SF-12. This controlled experiment confirmed the stability of the nanoparticles at low pH.The dual drug loaded TBN-CP-DOX also exhibited drug release profiles for cisplatin and DOX in the presence of esterase enzyme (see figure 4d). The triblock copolymer PEG-b-PCL50-bCPCL100 was subjected to biodegradation studies in the presence of esterase enzyme in PBS (pH = 7.4) at 37 °C for 24 h incubation. Aliquots were taken at regular intervals and the polymer samples were subjected to GPC. The GPC chromatograms (see SF 14)showed the decrease in molecular weights with increasing incubation time. The triblock copolymer has aliphatic polyester linkages which are biodegradable by esterase enzymes. Thus, individual drug loaded TBNs (TBN-CP and TBN-DOX) and dual drug loaded nanoparticle


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(TBN-CP-DOX) underwent identical enzymatic-biodegradation in the presence of lysosomal enzymes to release both DOX and Pt-drug.

Cytotoxicity and Synergistic Combination Therapy

Figure 5. Histogram depicting cytotoxicity of free cisplatin and TBN-CP in Wt MEF (a) and in MCF 7 cells (b). Histogram depicting cytotoxicity of free DOX and TBN-DOX in Wt MEF (c) and in MCF 7 cells (d). Histograms depicting cytotoxicity of free CP, free DOX and their cocktail (e). Histogram depicting cytotoxicity of dual loaded TBN-CP-DOX nanoparticles along with TBN-CP and TBN-DOX and their cocktail TBN-CP+TBN-DOX (f). Standard T Tests are also shown above. Two cell lines having large difference in the GSH content were chosen to test cisplatin drug delivery against GSH detoxification. Breast cancer cells (MCF 7) have 20 times over-expression of GSH (20 mM) compared to normal wild-type mouse embryonic fibroblasts (Wt MEFs, [GSH] = 0.5 to 1.0 mM).31-32 The cytotoxicity of the nascent TBN in Wt MEF and MCF 7 cell lines showed > 90 % cell viability confirming its high biocompatibility for drug delivery (see SF 15). In Wt MEF normal cells, free cisplatin drug exhibited > 90 % cell killing whereas the TBN-CP was found to be relatively less toxic 1.0). As shown in figure 6a, the free drugs were found to be antagonistic to each other whereas the individual drug loaded nanoparticles (TBN-CP plus TBN-DOX) exhibited additive effect. Interestingly, the CI values for dual drug loaded TBN-CP-DOX clearly demonstrated efficient synergistic cell killing in breast cancer cells. The synergistic effect by the dual drug loaded nanoparticles compared to individual drug loaded nanoparticles and free drugs is schematically shown in figure 6b. The enhanced combination therapy of TBN-CP-DOX in human breast cancer cells is attributed to both the inhibition of cell growth by DNA intercalation by DOX as well as delivery of cisplatin drug against the GSH detoxification from single polymer dose. Hence, the cisplatin conjugation and DOX encapsulation in TBN-CP-DOX provides unique opportunity in the present approach to enhance the breast cancer treatment efficacy.Though few efforts have been taken in the past to study the antogestic effect of cisplatin and DOX, a clear mechanism is yet to be understood.47 Cisplatin being an alkylating agent is known to bind to the N7 of the guanine base of double stranded DNA and result in interstrand crosslinks which induces apoptosis. Doxorubicin causes cytotoxicity by inhibiting topoisomerase II which creates a transient nick in the double stranded DNA and then catalyzes the process of re-ligation. This proceeds via formation of a DNA16 ACS Paragon Plus Environment

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topoisomerase II complex. Cells with over-expression of topoisomerase II become resistant to cisplatin since they bind to the cisplatin damaged DNA and initiate the DNA repair mechanism. Thus, the co-delivery of DOX and CP would overcome the problem mentioned before. Since DOX would poison topoisomerase II and thus it will not be able to repair the cisplatin damaged DNA, hence, resulting in a synergistic killing of cancer cells. This would only be possible when these drugs are delivered in a nanocarrier at the same site at a given time which brings them in close proximity, which is not the case in free drugs.

Cellular Uptake and mode of Apoptosis The cellular uptake of dual loaded TBNs in MCF 7 cells was monitored using confocal microscopy. The DOX fluorescence was observed in red channel and the nuclei were stained with DAPI (blue channel). In order to visualize the cell skeleton, the actin fibrils were stained with phalloidin and visualised through green channel.The images corresponding to DIC, DAPI, Phalloidin and DOX fluorescence are shown in figure 7a. The intensity of free DOX was relatively less compared to DOX delivered from TBN-DOX and TBN-CP-DOX. This strongly suggested that the DOX loaded TBNs efficiently deliver the drugs at the nucleus compared to free form. Flow cytometry of free DOX and TBN-DOX and TBN-CPDOX was carried out and their histograms are shown in figure 7b. The histogram clearly exhibited that the DOX internalization in MCF 7 cells was almost 4-fold higher for TBNDOX and TBN-CP-DOX compared to free DOX. The reason for the low uptake of free DOX (see Figure 7a) could be attributed to the fact that most of the cancer cells have MDRpumps48 that are well-known to efflux out the DOX. On the other hand the DOX loaded nanoparticles are larger in size and not possible to efflux them out from the cytoplasm. Thus, the drug loaded nanoparticles could be retained in the cytoplasm for longer duration and the slow release of drugs from TBN at the intracellular compartments enhanced the DOX concentration at the nucleus. The reason of the difference among the localization of TBN-CPDOX and TBN-DOX in Figure 7a was not clear at present. However, the flow cytometry analysis supports that both TBN-CP-DOX and TBN-DOX are taken in equal amount by the cells (Figure 7b). Thus, the triple block copolymer was capable of delivering the DOX almost in equal intensity from both TBN-CP-DOX and TBN-DOX. Thus, the newly designed TBN nanoparticles are capable of readily transporting both cisplatin and DOX across the cell membranes for synergistic cancer treatment.


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Figure 7. (a) CLSM images of MCF 7 cells incubated with DOX, TBN-DOX and TBN-CPDOX at 37 °C. (b) Flow cytometry plots for control, free DOX, TBN-DOX and TBN-CPDOX in MCF 7 cell line after 9 h incubation (DOX concentration = 1.84 µM and about 10,000 cells were used).

Figure 8. Apoptosis detection using Annexin V/PI flow cytometry assay of control (a), free CP (b), free DOX (c) and TBN-CP-DOX (d).


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The Annexin V flow cytometry assay detects the apoptotic cells and can distinguish between early apoptosis and late apoptosis or necrosis49 Annexin V is a Ca2+ dependent protein with high affinity towards phospholipid phosphatidylserine (PS) and thus it binds to the cells in the early apoptotic phase with exposed PS.49In addition to this, propidium iodide (PI) is used as a marker for necrotic cells since it is a membrane-impermeant dye and can only enter dead cells and bind to their DNA.50 The data for the Annexin V and PI analysis of control, free DOX, free CP, dual drug loaded TBN-CP-DOX are shown in figure 8. The untreated MCF 7 cells showed 86.7 % of healthy cell population along with a small fraction of cells that went through early apoptosis (0.74 %) and necrosis (6.04 %). In figure 9b, the free CP treated cells showed 4.45 % asearly apoptotic cells and 14.72 % cells for necrotic process. In free DOX treated cells, the early apoptotic and necrotic process were found to be 5.8 % and 21.4 %, respectively. The dual drug loaded TBN-CP-DOX treated cells exhibited 4.80 % of early apoptosis and 22.53 % necrotic cell death (see Figure 9d) which are almost comparable with that of free drugs. Based on this study, it can be concluded that the dual drugs released from TBN-CP-DOX nanoparticles were also found to follow the identical apoptosis and necrotic process as exhibited by the free drugs. These observations completely supported the fact that both the newly designed TBN platform was able to enhance the cisplatin and DOX uptake in breast cancer cells. This enhanced intracellular accumulation of cisplatin and DOX enumerates the fact that biodegradation of nanoparticles occurs upon exposure to the lysosomal conditions inside the cells. Through the TBN concept here is demonstrated exclusively for cisplatin along with DOX; in general, this approach an be expanded to wide ranges of other drug comnibation for better cancer therapy.

Conclusion In summary, the present work demonstrates unique biodegradable triple-block nano-carrier (TBN) geometry for the combination therapy of cisplatin and DOX. This design enabled the synergistic killing in cisplatin-resistant and GSH over expressed breast cancer cells. The TBN was designed having biocompatible PEG outer shell and biodegradable PCL and carboxylic PCL inner blocks for loading of anticancer drug DOX and conjugation of the cisplatin at the core. The dual drug loaded TBN-CP-DOX was found to be very stable in saline and PBS (> 90 %) and also stabilized the cisplatin drugs against the GSH detoxification at the cytoplasm. The biodegradable aliphatic PCL polyester backbone was responsive to lysosomal esterase enzymes and 19 ACS Paragon Plus Environment

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ruptured exclusively at the intracellular level to deliver the Pt-drug in active form. Cytotoxicity studies were done in Wt MEF, HeLa and MCF 7 cell lines. TBN-CP was found to be less toxic tot he normal cells (Wt MEF) compared to free cisplatin drug. In the GSH over expressed breast cancer cell line (MCF 7); the free cisplatin drug was detoxified and < 50 % of the cells were killed. The stable cisplatin conjugated triple layer TBN-CP exhibited > 95 % cell killing and this trend was attributed to the TBNresistance against drug detoxification by GSH. The combination index (CI) revealed that the cocktails of free drugs and cocktails of drug loaded nanoparticles (TBN-DOX and TBNCP) exhibited antagonistic and additive effect, respectively. The dual drug loaded single nanoparticle dose TBN-CP-DOX exhibited excellent synergistic cell killing at lower concentration of drugs through combination therapy. TBN-DOX and TBN-CP-DOX nanoparticles were found to accumulate in the cytoplasm and peri-nuclear site. Flow cytometry analysis revealed that the TBN-DOX and TBN-CP-DOX nanoparticles were more efficiently taken up by the cells as compared to free DOX. The present investigation established the proof-of-concept of co-delivery of cisplatin with DOX to achieve highly efficient “synergism” in cisplatin resistant breast cancer cells which could be extended to the other hydrophobic drugs too. Currently, the triple block nanoparticle strategy is being explored for various biomedical applications.

Supporting Information: Materials, methods, Synthetic details,

1

H-NMRs and GPC

chromatograms of the block copolymers. DLS data, FESEM and HRTEM images of polymer nanoparticles in water. FTIR, cumulative release profiles, cytotoxicity histograms of diblock copolymers. This material is available free of charge via Internet at http://pubs.acs.org. Acknowledgements: This work is funded by Department of Science and Technology (DST), New delhi, India under the project head SB/S1/OC-37/2013 and the authors thank DST for research funding.

Author

Information:

Corresponding

Author:

Prof.

Manickam

[[email protected]].The authors declare to financial interest.


Jayakannan

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Table of Contents

Triple Block Nanocarrier Platform for Synergistic Cancer Therapy of Antagonistic Drugs

Bapurao Surnar and Manickam Jayakannan*


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Triple Block Nanocarrier Platform for Synergistic Cancer Therapy of

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