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Targeted Smart pH and Thermoresponsive N,Ocarboxymethyl Chitosan Conjugated Nanogels for Enhanced Therapeutic Efficacy of DOX in MCF-7 Breast Cancer Cells Neeraj Kumar Verma, Mahaveer Prasad Purohit, Danish Equbal, Nitesh Dhiman, Amrita Singh, Aditya Kumar Kar, Jai Shankar, Sarita Tehlan, and Satyakam Patnaik Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.6b00366 • Publication Date (Web): 19 Sep 2016 Downloaded from http://pubs.acs.org on September 20, 2016

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Bioconjugate Chemistry

Targeted Smart pH and Thermoresponsive N,O-carboxymethyl Chitosan Conjugated Nanogels for Enhanced Therapeutic Efficacy of DOX in MCF-7 Breast Cancer Cells Neeraj K. Verma†,¥, Mahaveer P. Purohit†, ‡, Danish Equbal±, Nitesh Dhiman†, ‡, Amrita Singh† Aditya K. Kar†, ‡, Jai Shankar†, Sarita Tehlan∆ and Satyakam Patnaik†, ‡,* †

Water Analysis Laboratory, Nanotherapeutics & Nanomaterial Toxicology Group, CSIR-Indian

Institute of Toxicology Research (CSIR-IITR), Vishvigyan Bhawan, 31 Mahatma Gandhi Marg, Lucknow-226001, Uttar Pradesh, India. ¥

BBD University, College of Dental Sciences, Faizabad Road, Lucknow-226028, Uttar Pradesh,

India. ‡

Academy of Scientific and Innovative Research (AcSIR), CSIR-IITR Campus, Lucknow-

226001, Uttar Pradesh, India. ±

Medicinal and Process Chemistry Division, CSIR-Central Drug Research Institute (CSIR-

CDRI), Jankipuram Extension, Sitapur Road, Lucknow-226031, Uttar Pradesh, India. ∆

Department of Chemistry, Motilal Nehru College, University of Delhi South Campus, Benito

Juarez Road, South Campus, New Delhi-110021, Delhi, India. *

Author for correspondence: +918960420042

E-mail address: [email protected], [email protected]

ACS Paragon Plus Environment


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ABSTRACT In cancer treatment, developing ideal anticancer drug delivery systems to target tumor microenvironment by circumventing various physiological barriers still remains a daunting challenge. Here in our work, a series of pH and temperature responsive nanogels based on poly(N-isopropylacrylamide-co-2-acrylamido-2-methyl-1-propanesulfonate-co-1-propene-2-3dicarboxylate [poly-(NIPAAm-IA-AMPS)] cross-linked by ethylene glycol dimethacrylate (EGDMA) were synthesized by random copolymerization. The molar ratio between monomer:co-monomers:cross-linker was varied to fine-tune the optimum responsiveness of the nanogels. These optimized nanogels were further coupled to N,O-carboxymethyl chitosan (NOCC) stoichiometrically, using EDC-NHS coupling chemistry to enhance the swelling behavior at lower pH. Interestingly, these NOCC-g-nanogel when dispersed in aqueous media under sonication attain nanosize and retain their high water retention capacity with conspicuous pH and temperature responsiveness (viz. nanogel shrinkage in size beyond 35 0C and swelled at acidic pH) in-vitro as reflected by DLS data. Doxorubicin (DOX), a potent anticancer drug was loaded into these nanogels using physical entrapment method. These drug loaded nanogels exhibited a slow and sustained DOX release profile at physiological temperature and cytosolic pH. Further, confocal and TEM results demonstrate these nanogels were swiftly internalized by MCF-7 cells while cell viability data showed preferential heightened cytotoxicity towards cancer cells (MCF-7 and MDA-MB231) compared to the MCF10A cells (human breast epithelial cell). Furthermore, intracellular DNA damage, cell cycle arrest assays suggest a mitochondrial mediated apoptosis in MCF-7 cells. This study substantiates our NOCC-g-nanogel platform as an excellent modality for passive diffusive loading and targeted release of entrapped drug(s) at physiological conditions in a controlled way for improved therapeutic efficacy of the drug in anticancer treatment.


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Keywords: Nanogels, N,O-carboxymethyl chitosan, Apoptosis, Doxorubicin, Smart gels, Swelling and deswelling, Nanogels



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INTRODUCTION Several efforts have been made to develop target based drug delivery systems to treat diseases like cancer.

1, 2

But the premature release of the drug(s) from its carrier is still a major

bottleneck in drug delivery.

3, 4

Pre-release results in harmful toxic effects 5 as well as make the

drug unavailable for/to target cells, tissue or organs. Therefore, more efficient drug delivery strategies are the need of time. A possible way to overcome uncontrolled drug release is the use of stimuli-responsive materials which allow the drug to release in excellent spatial, temporal and dosage controlled fashion. Stimuli-responsive materials have the capability to recognize their microenvironment to respond the particular stimuli hence commonly termed as smart materials. Considerable progress in material science and pharmaceutics, particularly in the field of biomedical applications, has led to the development of novel stimuli-responsive nanocarriers as an efficient drug delivery system. 6 These stimuli-responsive nanocarriers are able to undergo a specific change such as protonation, hydrolytic cleavage or conformational change 7 in response to an endogenous and exogenous stimulus to the respective environment. Further, many stimuliresponsive nanocarriers have been used in the delivery of myriad therapeutic agent. 8 Among all the stimuli-responsive nanocarriers, nanogels are more promising carrier for cancer treatment. 9 Swelling/deswelling behavior of these nanocarriers further aid in developing newer pH and temperature responsive targeted nanogels.

10, 11

Recently, we reported a reagent

free route for synthesis of pH responsive polyacryloyl hydrazide (PAH) capped silver (Ag) or gold (Au) nanogels for anticancer therapeutic applications wherein, PAH possessing carbonyl hydrazide pendant functionality served as both reducing and capping agent to produce stable NPs.

12

Temperature and pH induced conformational changes of these nanogels also increased

the drug loading efficiency as well as limits their release to achieve better therapeutic efficacy. 13,


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14

Acidic microenvironment 15 and leaky vasculature of tumors have also been explored to design

pH and temperature responsive nanogels to target cancer cells. 16 Temperature sensitive polymers are gaining more attention in drug delivery because temperature stimulation may control the mesh size of the polymer matrix which is sine qua non for controlled release of entrapped drug. Poly N-isopropyl acrylamide (PNIPAAm), in particular, has been extensively studied and proven to be an indispensable component in many temperature responsive nanogels. PNIPAAm is also well-studied polymer for biomedical applications. 17-20 It shows volume phase transition (VPT) around 32 0C in water.

21

Heating above its lower critical

solution temperature (LCST) causes a rapid phase transition from hydrophilic to hydrophobic form, resulting in a collapsed state. Moreover, the acidic extracellular tumor microenvironment, as well as additional acidic intracellular endosomal environment, offers an avenue to explore this microenvironment as the sites for target-oriented drug release only upon pH stimulation. N,O-carboxymethyl chitosan (NOCC) is a well-known pH-responsive molecule, which is synthesized by partial substitution of carboxymethyl groups on pristine chitosan. NOCC is biocompatible, property,

24

22

biodegradable with high moisture retention capacity,

antibacterial and antifungal activity.

25, 26

23

have antioxidant

Many prior art substantiates the fact that

NOCC with variable molecular weight along with the presence of free carboxyl and amine groups render itself a chemically versatile molecule

27

and provides ample opportunities to the

researchers for developing newer strategies in drug delivery and tissue engineering applications. Moreover, numerous in-vitro studies on NOCC based drug delivery formulations such as hydrogels, nanoparticles and micelles have proven it to be non-toxic agent. 13, 28, 29 Protonation of the -NH2 groups (pKa - 6.5) at acidic environment makes it a suitable pH-responsive molecule for drug delivery in cancer treatment. 30-32



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In this study, a pH and temperature responsive NOCC modified PNIPAAm based nanogels (hereafter denoted as NOCC-g-nanogels) was developed by varying monomer to crosslinker ratio as a unique sustained drug delivery system. The discernible feature of this drug delivery system is : (i) its responsiveness to alteration in pH and temperature of its surrounding environment, (ii) high drug loading capacity, (iii) release of entrapped drug in slow and sustained manner, (iv) passive targeting of cancer microenvironment, and (v) ability to exert enhanced biological effect in-vitro. We achieved this by carefully choosing four components: NIPAAm, itaconic acid (IA), 2-acrylamido-2-methylpropane sulfonic acid (AMPS), and N, Ocarboxymethyl chitosan (NOCC). NIPAAm was used to achieve thermal responsiveness, IA moiety containing carboxylic group for subsequent tagging of dyes/drugs/antibodies etc., and AMPS used as a gelling component 33 and finally, NOCC was used to impart pH responsiveness to the nanogels. 34

RESULTS AND DISCUSSION Synthesis, Characterization and Morphological Analysis of Nanogels. Nanogels are synthesized utilizing simultaneous free-radical co-polymerization reaction.

35

APS was used as

an initiator to start the polymerization reaction. The major focus of this study was to design and synthesize a nanogel based delivery system which is (i) optimally tuned to respond to any alterations in temperature and pH of its surrounding environment, (ii) having the possibility of secondary modifications, and (iii) with high water retention capacity to allow passive diffusion of entrapped cargo upon hydration. To meet this challenge, we opted for NIPAAm as temperature responsive primary monomeric unit, IA as hydrophilic co-monomeric unit having free -COOH groups at both terminals for secondary conjugation of tagging probes and AMPS as a gelling component for water retention. Subsequently, a series of nanogels were synthesized by


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using fixed molar ratio of monomer to co-monomer and varying the cross-linker amount (see Table S1) which were then analyzed by DLS for their temperature responsiveness. Interestingly, out of six formulations of nanogels, only the nanogels synthesized with feed ratio 10:1 (monomer to cross-linker molar ratio), showed a decreasing trend in size with increase in temperature, whereas rest of the formulation showed a non-specific response to temperature increase. This was so because a higher degree of cross linking by the cross linker often render rigidness and stiffness to the gel network which in turn will restrict the VPT of the PNIPAm and insensitive to temperature responsiveness. Consistent with the earlier reported literatures that any subsequent addition of co-monomers to NIPAAm such as AMPS during polymerization process increases the LCST and the solvation energy of the copolymer leading to a reduction in swelling ratio, we observed a rise in the LCST temperature of the copolymer.

36, 37

As evident from the

LCST temperature of PNIPAm, below 32 0C these nanogels were in more hydrated form and remained in maximum swelling state having sizes in the range of 221 ± 3 nm at 25 0C, however, as the temperature rises above 37 0C, the nanogels collapsed shifting the equilibrium towards acquired hydrophobicity due to VPT, resulting in a more compactness of the gel networks and concurrently a decrease in size (198 ± 3 nm). It was also worthy to mention the decrease in PDI values (from 0.23 at 25 0C to 0.17 at 37 0C) indicative of the phase transition of these nanogels as shown in Table 1. Table 1: Temperature dependent size distribution of Nanogels (10:1; mol/mol) formulation. M:CL (10:1; mol/mol) Nanogel

Temperature (0C) 25

27

30

32

35

37

42

Size (nm)

221 ± 3

225 ± 2

219 ± 3

209 ± 2

207 ± 2

198 ± 3

202 ± 4

PDI

0.23

0.24

0.26

0.21

0.16

0.17

0.17

Zeta (mV)

-32

-31

-30

-30

-31

-30

-32



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It was also found the zeta potential of this (10:1; mol/mol) nanogel formulation has a high surface charge -30 to -32 mV across the temperature range indicating very high colloidal stability.

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Consequently, this nanogels formulation with optimally tuned properties is selected

for further subsequent studies. In order to impart amphiphilicity as well as to make these nanogels target specific and pH responsive, NOCC was covalently conjugated to the free carboxyl groups of the IA segment using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) mediated coupling chemistry. It is well known that NOCC is a safe and sensitive biocompatible material that responds to slight pH change around the physiological pH of 7.4. The pH responsiveness of NOCC is due to swelling induced protonation of amino-groups (pKa-6.3).39 The degree of NOCC conjugation to these nanogels was further investigated to ensure that they retain their desired temperature responsiveness while showing an additional response to pH alterations. For this, the ratio of NOCC to nanogels was varied stoichiometrically (1:1, 2:1, 5:1, w/w) and studied for change in hydrodynamic diameter in response to pH and temperature by DLS.

It was found that out of three formulation ratios of the NOCC-g-nanogels, (1:1, w/w) formulation has the desired characteristics with optimum size in response to temperature (37 0C) and acidic pH (5.4) as shown in Table 2 while the rest of the two formulations did not show any particular trend (see Table S2). The increase in size and PDI values of these NOCC-g-nanogels compared to the nanogels only (from 221 ± 3 to 300 ± 2.4 nm at 25 0C and from 0.23 to 0.35 mV, respectively) clearly support the successful conjugation process. An inverse correlation was observed between the rise in temperature and decrease in pH on the size of the NOCC-gnanogels synthesized with 1:1 formulation ratio. Thus, this nanogel formulation was used for further subsequent studies. It was envisioned that the interplay of forces responsible for swelling


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and deswelling of these NOCC-g-nanogels at elevated temperature and lysosomal acidic pH respectively, will dictate the release kinetics of the entrapped drug molecules over a sustained period. Table 2:Temperature and pH dependent size distribution of NOCC-g-nanogels (1:1, w/w) formulation. NOCC-gnanogels (1:1, w/w)

Temperature (0C)

pH 3.0 pH 5.5 (at 25 0C) (at 25 0C)

pH 7.4 (at 25 0C)

25

30

32

35

37

Size (nm)

300 ± 2.4

283 ± 6

261 ± 2.4

241 ± 2

237 ± 2.1

574 ± 2.8

334 ± 2

307 ± 1.3

PDI

0.35

0.41

0.33

0.32

0.33

0.65

0.23

0.33

Zeta (mV)

-20 ± 0.8

-18 ± 0.6

-21 ± 1.4

-19 ± 0.4

-17 ± 1.0

-6

-11

-22

FTIR Analyses. Figure 1 A, shows the FT-IR spectra of NOCC, nanogels, and NOCC-gnanogels. The spectrum of NOCC showed a broad -OH stretch absorption band between 3600 and 3100 cm-1 and the aliphatic C-H stretch at around 2883 cm-1. As the -OH stretch band and the aliphatic C-H stretch band are aligned, it appears as a broad band from 3600 and 2883 cm-1 in the spectrum. Another major absorption band at 1235 cm-1 represents the free primary amino group (-NH2) at C2 position. The peak at 1625 cm-1 represents an acetylated amino group of NOCC, which indicates that the sample is not fully deacetylated. It was also observed in the spectrum of NOCC peaks at 1521 and 1378 cm-1 characteristic of asymmetry and symmetry stretching vibration of COO- respectively, suggesting that there were carboxymethyl groups existing on NOCC. Also, the C-O adsorption peak of the secondary hydroxyl group appears as a stronger band at 1065 cm-1 (Figure 1A (a)). In nanogel spectrum (Figure 1A (b)), the characteristic peaks of amide I, II and primary amine are observed at 1640, 1539 and 3288 cm-1 respectively. Further, peaks at 1040 and 1174 cm-1 corresponding to vibration bands of sulfonic



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groups does indicate the incorporation of AMPS component in the nanogel after polymerization. In addition, the characteristic peaks of NIPAAm monomer at 2975, 1458 and 1388 cm-1 assigned to CH, C=C, and CH2, respectively has shifted to 2933, 1455 and 1368 cm-1 in NOCC-gnanogels confirming successful integration. Likewise, the spectrum of NOCC-g-nanogels (Figure 1A (c)) have peaks due to the methyl group of -CH(CH3)2 of PNIPAAm appeared around 1387 cm-1 and those due to COO- of NOCC were observed around 1631 cm-1. Moreover, the peaks at 1065 and 1039 cm-1 corresponding to NOCC and nanogels respectively do appear in NOCC-g-nanogels at 1066 and 1037 cm-1 also. 1H NMR spectrum of equilibrium swollen poly(NIPAAm-IA-AMPS nanogels gels in D2O in the ‘‘liquid mode’’ at room temperature (25 0C) is shown in Figures S2 (A). The spectrum has adequate resolution to identify six distinct peaks of the polymer chain protons and a high peak of the HOD proton. The chemical shifts of these peaks are 1.02, 1.17, 1.36, 1.99, 2.59, 3.77 and 4.5 ppm in reference to TMS respectively. Moreover, the appearance of broad peaks at δ 3.77 (lone proton of the N-isopropyl group of NIPAAm; –H

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