Engineering a New Class of Multiarm Homopolymer for Sustainable

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Engineering New Class of Multi-arm Homopolymer for Stimuli Responsive Drug Delivery Mutyala Naidu Ganivada, Pawan Kumar, Ajin Babu, Jayasri Das Sarma, and Raja Shunmugam ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.7b00100 • Publication Date (Web): 01 Apr 2017 Downloaded from http://pubs.acs.org on April 6, 2017

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ACS Biomaterials Science & Engineering

Engineering New Class of Multi-arm Homopolymer for Sustainable Drug Delivery Mutyala Naidu Ganivada1, Pawan Kumar1, Ajin Babu1, Jayasri Das Sarma2# and Raja Shunmugam1* 1

Polymer Research Centre, Department of Chemical Sciences, 2 Department of Biological Sciences, Indian Institute of Science Education and Research Kolkata Mohanpur Campus, Mohanpur, Nadia, West Bengal – 741246, India. Email: [email protected]

Abstract: The first example of simple nano-aggregates, self-assembled from a new class of polymer has been explored. The synthesis and characterization of polyethylene glycol and doxorubicin attached to 1,6-heptadiyne derivative (Macromonomer) are clearly described. The Macromonomer is polymerised via olefin metathesis living cyclopolymerization method using Grubbs-Hoveyda catalyst to produce a water soluble Dox-Peg-Rcp-Fmoc polymer. All the monomers and polymer are carefully characterised by GPC and 1H NMR spectroscopy. Fmoc deprotection of Dox-Peg-Rcp-Fmoc polymer is carried out to produce Dox-Peg-Rcp polymer. The newly designed Dox-Peg-Rcp polymer shows nano-aggregation in water. In addition to that, they are soluble in water as well as biological medium. The drug release profile at mild acidic condition shows the importance of having ester linker. CLSM images of Dox-Peg-Rcp nano-aggregates clearly show the efficient internalisation into the living cells. MTT assays of Dox-Peg-Rcp nano-aggregates show that these nano-aggregates have a greater anticancer efficacy. To best of our knowledge, this is the first report on water soluble polyacetylenes for the biological/drug delivery application. Keywords: RCMP, Hoveyda Grubbs catalyst, polyacetylenes, self-assembly, doxorubicin.

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Introduction: In recent years, polymer-based nanostructures are most promising for biomedical applications.1,2 Polymer-drug conjugates provide promising hopes in cancer treatment with defined biological rationale.3-5 Morphology of the polymer nanostructures plays a key role in drug delivery.6 Polymer nanostructures, due to self-assembly, show potentials in ‘enhanced permeation and retention’ (EPR) effect.7 Living polymerization gives control over molecular weight, size and morphology of the polymer, but limited range of organic functional groups. So, development of new class of polymer is always a trend for therapeutics. The idea of the copolymer has been introduced for the synthesis of conjugated copolymers to increase the solubility, to demonstrate unique optical properties and morphology control.8 These copolymers are synthesised by either ‘grafting from approach’9-12 or ‘grafting through approach’.13-16 But there are only a few examples are reported for the direct synthesis of conjugated polymer by the grafting through approach due to the synthetic difficulties arising from steric hindrance. Nowadays, ring-opening metathesis polymerisation (ROMP) has been employed as one of the best method for the synthesis of polymers by the grafting through approach.17-21 But it is only applicable for norbornenyl macromonomers using Grubbs’ catalyst. Polymerization of 1,6 heptadiyne by Ring Closing Metathesis Polymerization (RCMP) has attracted researchers recently due to its unique way of generating conjugated polymers with multiple functionalities.22 This polymerization also undergoes living fashion with narrow polydispersity index. In literature, many polymer drugs conjugated for the cancer therapy using different polymerization techniques are reported.23-27 But these polymers have limitations for attachment of non-fluorescent drug molecule without any fluorescent probe.28 Polyacetylenes are very active in the UV-Vis region due to conjugation in the backbone. Due to its optical property polyacetylenes can synthesise and functionalize for drug conjugates


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which utilises the optical properties of polyacetylenes to monitor the drug release in cancerous cells without attachment of fluorescent probe. The cyclopolymerization is the best method for the synthesis of substituted polyacetylenes via olefin metathesis. The first example of the living cyclopolymerization of 1,6-heptadiyne derivatives have been reported by the Schrock and co-workers.29,30 Buchmeiser and coworkers

have

reported

Mo

alkylidene

with

quinuclidin

catalyzed

selective

cyclopolymerization, to give conjugated polymers with five - membered ring structure in living manner.31-33 Recently, Tae-Lim Choi and co-worker have reported semiconducting poly(L-Lactide) and poly(caprolactone) via olefin metathesis cyclopolymerization method using Grubbs-Hoveyda catalyst.34 Nevertheless, a major drawback of polyacetylenes polymers is its hydrophobicity. For the biological application, the polymers should be water soluble. In recent years several methods and procedures have been reported to control release rate of drugs by attaching them covalently to an acid labile bonds like imine,35 acetal,36 oxime,37-39 hydrazone,40 orthoester.41 Among these acid labile bonds, we are very specific about ester linker as it releases the drug under low acidic conditions, which resembles the cancerous cells pH. Multi-arm polymeric micelles have shown superior potential and have gained more attention in comparison to conventional polymeric micelles due to its enhanced prolong therapeutic efficacy and triggered release influencing controlled and selective drug delivery responses with reduced side effects.42-45 Herein, we report the synthesis of polyethylene glycol and doxorubicin attached multi-arm 1,6-heptadiyne derivative (Macromonomer) and its polymerisation via olefin metathesis living cyclopolymerisation method using GrubbsHoveyda catalyst to produce water soluble Dox-Peg-Rcp-Fmoc polymer for anti-cancer agent. The newly designed amphiphilic Dox-Peg-Rcp polymer forms nano-aggregates in aqueous environment. The drug release profile indicates the advantage of having the ester



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linker. CLSM images of Dox-Peg-Rcp show that nano-aggregates are effectively internalized by living cells. MTT assays of Dox-Peg-Rcp nano-aggregates show that these nanoaggregates posses a significant anticancer efficacy. To best of our knowledge this is the first report with water soluble polyacetylenes for the biological application. We believe that the newly designed polymers will set up a new platform in the field of drug delivery. The main advantage of the polyacetylenes is their red-orange nature due to the conjugation in the backbone. This will be a huge advantage when the delivery of non-fluorescent drug molecules, for example, chlorombucil, cis-platin, etc. are attached to this backbone. Exploring the wide application of polyactetylene backbone on the drug molecules, which are neither having color nor fluorescence, would be our future report. Results and Discussions: Towards the motivation of synthesizing water soluble polyacetylenes, we started the synthesis of mPeg-NH2 using previously reported procedure46,47 as shown in Scheme S1. Fmoc protected doxorubicin (compound 4) was prepared by our previously reported procedure48 as shown in Scheme S1 (Figure 1b). After that, we started to prepare Macromonomer as shown in Scheme 1. First, compound 5 was synthesised by treating malonic ester with propargyl bromide using our previously reported procedure49 (Figure S1 & S2). Compound 6 was synthesized by the selective monoester hydrolysis of compound 5 with KOH and dry ethanol. In 1H NMR spectrum (Figure S3) of compound 6, a new peak was appeared at δ 8.4 ppm which was due to a carboxylic acid proton, suggested the formation of compound 6. Next, compound 7 was prepared by the coupling reaction between N-hydroxy succinamide and compound 6. In 1H NMR spectrum (Figure S5), the disappearance of carboxylic acid proton peak at δ 8.4 ppm and appearance of a new peak at δ 3.1 ppm, which was responsible for N-hydroxy succinamide proton, confirmed the formation of compound 7. Compound 8 was prepared by


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treating of compound 7 with compound 3 in presence of triethylamine and dimethylformamide. The absence of the peak at δ 3.2 ppm corresponded to Nhydroxysuccinimide and appearance of a new peak in the region δ 3.5 ppm which was responsible for an mPeg-NH2 proton in 1H NMR spectrum (Figure S7) which confirms the formation of compound 8.



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O

O

O NaH. 0-5

O

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O

O

oC

KOH O

O

O

O

Dry THF

O OH

Anhydrous Ethanol

Br

6

5 EDC-HCl HOBT Dry DMF O TEA, Dry DMF

H N

O O

8

O

O

O

H2N

43

O

O 7

LiOH Anhydrous Ethanol O H N O

O

OH OH

43

O

OH

+

O

O

O

O HO O

H O

9 Fmoc N H

EDC-HCl HOBT Dry DMF

O

O

O HO O

H N

O OH

O

O

H O

Fmoc N H

OH 4

OH

OH

10 (Macromonomer)

Scheme 1: Synthesis of Macromonomer.


O N

O 43

O 3

HO

O

O

O

O 43

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Scheme 2: Synthesis of Dox-Peg-Rcp-Fmoc and Dox-Peg-Rcp polymers. Next, compound 9 was prepared by the ester hydrolysis of compound 8 with weak base (lithium hydroxide). In 1H NMR spectrum (Figure S9), the peaks at δ 4.2 and 1.2 ppm were completely disappeared which confirmed the complete deprotection of ester group from compound 8. All the compounds (compound 5, 6, 7, 8 & 9) were further confirmed by

13

C

NMR (Figure S4, S6 & S8), FT-IR and MASS spectroscopies. Then, the Macromonomer was prepared by the coupling reaction between compound 9 and compound 4 (Fmoc-Dox) with DCC and DMAP. The formation of product was confirmed through NMR and FT-IR spectroscopic techniques. In 1H NMR spectra of the Macromonomer showed the signals of the aromatic protons of doxorubicin and Fmoc group (δ 8.00-7.00 ppm) along with all other doxorubicin peaks (Figure 2a).



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Figure 1: a) Comparison study of FT-IR spectrum for synthesis of Peg-NH2. b) 1H NMR of Fmoc-Dox in Jeol 400 MHz NMR. The signals of the Peg spacer of the Macromonomer (δ 3.70-3.40 ppm) confirmed the Macromonomer formation. In addition, the molecular weight of newly synthesised Macromonomer was obtained as Mn = 2,800 g/mol with narrow PDI = 1.03 (Figure 2b) by GPC using THF as eluent, which confirmed the Macromonomer formation.


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Figure 2: a) 1H NMR spectrum of Macromonomer. b) GPC study of i) Macromonomer ii) Dox-Peg-Rcp-Fmoc polymer iii) Dox-Peg-Rcp polymer. After the successful synthesis of Macromonomer and thorough characterization by different spectroscopic techniques, the polymerisation of Macromonomer was executed using Hoveyda-Grubbs 2nd generation catalyst (Scheme 2). After 2 hours, resultant reaction mixture was quenched by ethyl vinyl ether and followed by precipitation in diethyl ether. In



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1

H NMR spectrum, the new peaks were appeared at δ 5.2 to 5.5 ppm and peak for acetylene

protons at δ 2.1 ppm completely disappeared, which suggested the formation of polymer.

Figure 3: a) DLS study of Dox-Peg-Rcp polymer in water. b) FE-SEM images of Dox-PegRcp polymer in water. In addition, the molecular weight of Dox-Peg-Rcp-Fmoc polymer was estimated by GPC instrument. The GPC trace of Dox-Peg-Rcp-Fmoc polymer was observed as unimodal with Mn = 13,000 g/mol and PDI = 1.23 (Figure 2b). According to the GPC, the shifting of GPC trace of Macromonomer from high retention time to low retention time (Figure 2b) indicated the formation of polymer. Fmoc deprotection of Dox-Peg-Rcp-Fmoc polymer was carried out in presence of pipyridine to produce Dox-Peg-Rcp polymer. After the deprotection of Fmoc group, the molecular weight of polymer obtained from GPC was decreased to 12,200


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g/mol with 1.22 PDI (Figure 2b). In addition to that, the disappearance of Fmoc group proton signals in 1H NMR spectrum confirmed the formation of final Dox-Peg-Rcp polymer (Figure S10). After successful design and synthesis of Dox-Peg-Rcp polymer, Due to amphiphilicity in the polymer, we measured the critical aggregation concentration (CAC) by fluorescence intensity of Nile red at 636 nm with different polymer concentration. Fluorescence intensity was almost same up to a certain polymer concentration. Then there was significant change was observed in the fluorescence intensity due to the dye encapsulation in the polymer aggregates. Then CAC was calculated between fluorescence intensity of Nile red vs. Polymer concentration. The observed CAC was 1.5 µg/ml (Figure S13). To measure the size of the micelles we carried out using DLS analysis under aqueous environment. For this, 1 mg of Dox-Peg-Rcp polymer was solubilised in 10 ml of HPLC water and self-assembly study was done. From the above solution, an aliquot of the solution was taken to study the particle size using DLS. From DLS (Figure 3a), the observed size of nano-aggregates was 80 nm. Further, the same solution was taken to study the morphology of Dox-Peg-Rcp polymer using FE-SEM. The solution was cast on a silicon wafer and it was dried completely under the reduced pressure. From FE-SEM study (Figure 3b), the size of the nano-aggregates was mastered around 70 nm, which was in good agreement with DLS data. The observed shape of the nano-aggregates was capsule-like structure. It was interesting to observe the capsule-like self-assembled structures because these types of shapes are always preferred over the conventional spherical type shape due to their better blood circulation capabilities. Since, the pH of normal cells in human life is 7.4 and endosomes, lysosomes are acidic in nature, the drug release study of Dox-Peg-Rcp polymer was examined at pH 7.4 as well as pH 5.5 in phosphate buffer solution through UV and fluorescent spectroscopy.



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Figure 4: a) Drug release study of Dox-Peg-Rcp polymer at 37 °C at pH 5.5 and 7.4. b) Cytotoxicity profile of Dox-Peg-Rcp polymer in HeLa wt cells (human cervical cancer cell line). Towards this goal, 1 mg of Dox-Peg-Rcp polymer was solubilised in 1 mL of deionized water and subsequently loaded into 3,500 Dalton cut-off dialysis tube and dialysed in 100 mL of phosphate buffer solution with pH 5.5. From this solution, an aliquot was taken and


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measured for the release of doxorubicin from the Dox-Peg-Rcp polymer by measuring its absorbance at 480 nm by UV spectroscopy (Figure S11). Fluorescent was also studied by exciting the solution at 510 nm and the emission was observed at 560 nm and 590 nm (Figure S12), which indicated the release of doxorubicin from Dox-Peg-Rcp polymer. The aliquot was again added back to the solution to keep the constant volume of the solution. This process was repeated at an interval of 1 hour for 24 hours. After 12 hours, there were no significant changes in the intensity of fluorescence spectroscopy. The similar experimental procedure was carried out for the drug release at pH 7.4. It was interesting to observe that the micelles’ of Dox-Peg-Rcp polymer was stable at physiological condition i.e. pH 7.4, because of minimal release (10%) of doxorubicin from Dox-Peg-Rcp polymer (Figure 4a). It was also observed that at pH 5.5 release of doxorubicin from Dox-Peg-Rcp polymer was around 30% in first 1 hour because of burst phase and followed by a slower release in next 1 hour with 15% and 10% respectively. Finally, the maximum release of doxorubicin was around 75% after 12 hours. So, it was interesting to observe the importance of ester linker with DoxPeg-Rcp polymer. After successfully studied the drug release study at different pH, in vitro study with MTT assay was explored. Cellular uptake and cellular imaging were studied using CLSM. Cells were treated with Dox-Peg-Rcp polymer with concentrations of 25 µg/ml to 500 µg/ml for 24 hours, 48 hours and 72 hours respectively. Cytotoxicity assay measured by MTT. A fresh 20 µl (from stock concentration 5 µg/ml) solution of MTT was added to each well, followed by incubation for 4 hours in 5% CO2 at 37 °C and media was replaced by 100 µl isopropanol from each well of 96 well plate. Purple colored solution was measured by ELISA Plate Reader. From, the cell viability assay (Figure 4b) it was clear that when the concentration of the Dox-Peg-Rcp polymer nano-aggregates was high and later time period the efficiency was



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more compared to low dose of Dox-Peg-Rcp polymer and early time point. The maximum lethal dose was recognizing in 250 µg/ml and 500 µg/ml in 72 hours.


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Figure 5: a) CLSM images of Dox-Peg-Rcp in HeLa wt cells (human cervical cancer cell line) b) Pictorial representation of proposed mechanism of our design. For Dox-Peg-Rcp polymer nano-aggregates cellular uptake study was done in (cervical cancer) HeLa wt cells. For cellular imaging analysis, cells were seeded with a cover slip in 24 well plate at a density of 1.5 × 105 cells per well for 24 hours in the minimum essential medium. Cells were treated with Dox-Peg-Rcp polymer nano-aggregates with different drug concentration (25, 50, 100 µg/ml) at 37 °C. After 24 hours and incubation, coverslips were washed with 1 x PBS for three times, and cells were fixed in 4% paraformaldehyde (PFA) for 10 min. After cell fixation with PFA, cells were washed with 1X PBS for five times to overcome any background noise. From the cellular uptake studies (Figure 5a) it was clear that the nano-aggregates internalised very effectively with a higher concentration of nanoaggregates. Conclusion: This paper described a new class of polymeric pro-drug micelle for the cancer therapy via olefin metathesis living cyclopolymerisation technique (RCMP). The polymeric micelle showed properties like well-shielded drug moieties, significant water solvable, explicit nanostructure and low pH acid-triggered drug release to increase the efficacy of loaded drugs. Self-assembly nature of newly designed amphiphilic Dox-Peg-Rcp polymer was confirmed by DLS and FE-SEM analysis. MTT assay of Dox-Peg-Rcp polymer clearly showed that the efficiency was excellent at higher concentration of the Dox-Peg-Rcp polymer nanoaggregates also with longer time period compared to the lower dose of Dox-Peg-Rcp polymer nano-aggregates. Confocal laser scanning microscopy (CLSM) of Dox-Peg-Rcp polymer nano-aggregates on HeLa wt cells confirmed that very efficient cellular uptake was observed with higher concentration of polymer than the low concentration of polymer. Supporting Information:



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Experimental Section, synthesis scheme of all compounds, 1H NMR,

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C NMR, UV,

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Anders,

U.;

Nuyken,

O.;

Buchmeiser,

M.

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Engineering New Class of Multi-arm Homopolymer for Sustainable Drug Delivery Mutyala Naidu Ganivada1, Pawan Kumar1, Ajin Babu1, Jayasri Das Sarma2# and Raja Shunmugam1* 1

Polymer Research Centre, Department of Chemical Sciences, 2 Department of Biological Sciences, Indian Institute of Science Education and Research Kolkata. E-mail: [email protected]


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Engineering a New Class of Multiarm Homopolymer for Sustainable

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