pH and thermosensitive 5-fluorouracil loaded poly(NIPAM-co-AAc) nanogels for cancer therapy

Abstract

The aim of the study was to develop 5-FU loaded pH and thermo-sensitive nanogels, capable of sensing change in tumour environment pH and triggering drug release at physiological temperature. Poly(N-isopropylacrylamide-co-acrylic acid) (PNA) nanogels (Ng's) were prepared via free radical polymerization. The prepared nanogels were screened for FT-IR, ¹H NMR, pH dependent lower critical solution temperature (LCST), and DLS. Selected Ng's were loaded with 5-FU and screened for FTIR, particle size, TEM, % EE, in vitro drug release, % hemolysis, pH dependent cytotoxicity, in vivo kinetics on Wistar rats, in vivo anti-cancer efficacy and mean life span on Swiss albino mice. FTIR and ¹H NMR confirmed the formation of PNA. The prepared nanogels exhibited pH dependent LCST. The particle size of selected PNA and 5-FU–PNA Ng's were found to be 70.25 and 131.5 nm respectively at 25 °C. Drug release from 5-FU–PNA Ng's was found to be extended and highly sustained at pH 5.5, 6.8 and 7.4. However, when compared with respect to individual pH, drug release in pH 7.4 PBS was quite minimal when compared to pH 5.5 and 6.8. The cytotoxicity of 5-FU–PNA Ng's increased with decrease in pH value. Additionally they could extend in vivo circulation time and displayed effective anti-cancer efficacy when compared to 5-FU injection for the same. Our results documented enhanced anti-cancer efficacy and mean life span in EAC bearing mice.

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1. Introduction

Even though the advances in treatment and management of cancer have progressed over the years, its complete eradication still remains a conundrum for researchers as well as physicians. According to the WHO cancer report 2014, cancer still remains a leading cause of morbidity and is responsible for at least 8.2 million deaths each year and is accountable for 14 million new cases each year. Research suggests that the number of new cases is expected to rise by approximately 70% in the upcoming two decades.¹

5-Fluorouracil (5-FU), a fluorinated analog of the pyrimidine uracil, is well known for its anti-metabolite and anti-neoplastic actions and applications in the treatment of different types of cancer. Principally, it acts as a thymidylate synthase (TS) inhibitor via blocking the enzyme synthesis of pyrimidine thymidine, a nucleoside required for DNA replication. Consequently, inhibition of TS leads to a scarcity of thymidine monophosphate (dTMP) that ultimately causes cell death.² A major drawback of 5-FU and current chemotherapeutic regimens has been associated with severe side effects such as cardiac, lung, liver, and nephrotoxicity which also affect emotional status of the individual and higher risk of relapse.^3–6 In order to address the aforementioned problems, extensive research on developing targeted drug delivery systems for existing molecules as well as synthesis of new potential anti-neoplastic/anti-metabolites candidates is being carried out.^7–11 However, a complete breakthrough for successful treatment still remains a dilemma.

More recently altered tumour microenvironment and pH has generated substantial interest in developing pH responsive drug delivery systems.^12–15 It is well known that extracellular nature of tumour cells/tissues is acidic in contrast to physiological pH of healthy cells that are basic.^16–19 Therefore, difference in pH could be used as a tool to trigger the release of drug at the tissue/cell of interest.^16,20 Thermo-sensitive polymers that are hydrophilic in nature but become hydrophobic beyond its lower critical solution temperature (LCST) can be used as a secondary stimuli for triggering the drug release.^21,22 Therefore, a system responding to more than one stimuli would be a boon to the existing mono stimulus systems.²³ pH and thermo-sensitive nanocarriers that could alter its LCST, based on the difference in micro-environmental pH, could be utilized as an effective carrier for targeting the drug.²⁴ Amongst the available pH and thermo-sensitive polymer, poly(N-isopropylacrylamide-co-acrylic acid) (PNA) has been extensively reported for its pH dependent thermo-sensitivity property and its application for targeting drugs to the site of choice.^25,26

Previously Xiong et al.,²⁷ made an attempt to prepare pH and thermo-sensitive PNA nanoparticles covalently conjugated with doxorubicin and studied its potential application in tumour hyperthermia treatment. Sanoj Rejinold et al., attempted to develop 5-FU loaded pH and thermo-sensitive chitosan-graft-poly(N-isopropylacrylamide)²⁸ and fibrinogen-graft-poly(N-isopropylacrylamide)²⁹ and studied their application for breast cancer therapy using MCF-7 breast cancer cells. Even through the respective studies highlighted the applications of pH and thermo sensitive over cancer therapy, there is no sufficient in vivo anti-cancer efficacy studies highlighting their application. In the present study, an attempt has been made to prepare self regulated pH and thermosensitive PNA Ng's; capable of sensing the change in tumour environmental pH and trigger the drug release at physiological temperature. 5-FU was selected as a model drug loaded into selected PNA Ng's and studied for its in vitro drug release, cytotoxicity, in vivo kinetics, in vivo anticancer efficacy and mean life span.

2. Materials and methods

2.1. Materials

5-FU was a gift sample obtained from Sigma-Aldrich, India. N-Isopropylacrylamide (NIPAM) was procured from Tokyo Chemical Industry Co., Ltd. Tokyo, Japan. Acrylic acid (AAc) and sodium dodecyl sulphate (SDS) were procured from Loba Chemicals, Mumbai. Potassium peroxodisulphate (KPS) was procured from Merck, Mumbai. N,N′-Methylenebis(acrylamide) was procured from Sigma Aldrich, USA. NIPAM was purified by re-crystallization in methanol/n-hexane (50 : 50). All other excipients and chemicals used were of analytical grades.

2.2. Development of poly(N-isopropylacrylamide-co-acrylic acid) nanogels

Poly(N-isopropylacrylamide-co-acrylic acid) Ng's were prepared using free radical polymerization. As listed in Table 1, a series of nanogels were prepared by varying the monomer feed composition (NIPAM and AAc).^27,30 The total monomer in feed was kept constant at 0.015 mol/100 mL and feed composition ratios were varied accordingly (98 − x : x : 2; NIPAM : AAc : MBA). The monomers, crosslinker MBA (46 mg) and surfactant SDS (58 mg) were initially dissolved in 95 mL of distilled water. The prepared solution was heated at 70 ± 5 °C for 1 h and constantly stirred at 300 rpm using magnetic stirrer. Polymerization was initiated by addition of 5 mL distilled water containing 17 mg KPS. Nitrogen was purged for 15 min prior to addition of initiator. The reaction process was continued for next 4 h with above set parameters. Polymerization was terminated by reducing the temperature of the solution to room temperature. The resulting solution was dialysed for 5 days at room temperature and further freeze dried using Christ Alpha 2-4 LD Freeze Dryer. % practical yield was determined gravimetrically (eqn (1)).

Table 1 Feed composition ratio of prepared PNA nanogels

Code	Feed composition ratio^a (NIPAM : AAc : MBA)	% practical yield	Obtained polymer composition^b mol%
			NIPAM	AAc
a Total monomer in feed was 0.015 mol. Feed composition ratio's used (98 − x : x : 2).b Obtained polymer composition (mol%) was calculated using ^1H-NMR results.
PNA0	98 : 0 : 2	83.45	100	0.00
PNA1	97 : 1 : 2	80.25	99.05	0.95
PNA1.5	96.5 : 1.5 : 2	87.23	99.36	0.64
PNA2	96 : 2 : 2	79.07	97.16	2.84
PNA2.5	95.5 : 2.5 : 2	78.31	91.18	8.82

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2.3. Characterization and evaluation of poly(N-isopropylacrylamide-co-acrylic acid) nanogels

The nanogels were screened for Fourier transform infrared spectroscopy (FT-IR), proton nuclear magnetic resonance (¹H NMR), pH dependent lower critical solution temperature (LCST) and change in hydrodynamic diameters in response to pH and temperature.

2.3.1. Fourier transform infrared spectroscopy (FT-IR). FT-IR spectral analysis was carried-out by employing sodium chloride (NaCl) cell using FT-IR spectrometer, FTIR 8400-S, Shimadzu. Test samples were prepared by dispersing small amount of freeze dried nanogels/NIPAM/AAc/MBA in dichloromethane. The prepared sample was mounted on NaCl cell and subjected for FT-IR spectral analysis.

2.3.2. Proton nuclear magnetic resonance (¹H NMR). D₂O swollen nanogels were characterized for ¹H NMR using Bruker-400. D₂O swollen nanogels were prepared by dispersing 10 mg of freeze dried nanogels in 5 mL of D₂O and allowed to swell overnight.^30,31

Polymer composition was calculated by comparing peak area of NIPAM C–H at 3.9 with total peak area between 0.7 and 2.2 ppm. Further, % copolymer composition was calculated by comparing ratio of copolymer with that of homopolymer.

2.3.3. pH dependent lower critical solution temperature (LCST). pH dependent LCST of prepared nanogels were evaluated by measuring the change in absorbance at 500 nm using UV spectrophotometer (UV1800, Shimadzu). 1 mg mL⁻¹ freeze-dried nanogels suspension was prepared using saline and phosphate buffer solution (PBS) of different pH i.e., pH 5.5, 6.8 and 7.4 respectively. The prepared nano-suspensions were allowed to swell overnight at 10 °C. Post swelling, the prepared sample solution temperature was ramped up from 10 °C to 50 °C. LCST of nanogels was defined as the temperature showing raise in 0.5 absorbance (Abs).³²

2.3.4. Change in hydrodynamic diameters in response to pH and temperature. Nanogels which exhibited desired LCST were evaluated for change in hydrodynamic diameters in response to pH and temperature using dynamic light scattering (DLS) technique (Zetasizer, Nano ZS90™, Malvern instruments) equipped with He–Ne laser at λ_max 633 nm.³³ The samples were prepared as discussed above, and equilibrated for 60 s at each temperature interval.

2.4. 5-FU loaded PNA Ng's

2.4.1. Preparation and evaluation of 5-FU loaded PNA Ng's. Selected PNA Ng's were loaded with 5-FU by dispersing 2 g of placebo PNA Ng's into 100 mL of distilled water containing 500 mg of dissolved 5-FU. Sample solution was incubated for next 48 h at refrigerating conditions (4 °C). Post 48 h, sample solution was dialysed against known amount of distilled water (900 mL) in order to remove free 5-FU from the solution. Dialysis was carried out for a period of 4 h at ambient temperature (ambient temperature of distilled water was 24 ± 2 °C at the time of study). Amount of 5-FU in dialyzed fluid was determined by measuring the absorbance at 265 nm using UV spectrometer.

Entrapment efficacy (% EE) was determined using following eqn (2).

where “mo” is actual drug dosage taken, “c” is concentration in dialysis fluid and “V” is volume of dialysate fluid.

The resulting 5-FU loaded PNA (5-FU–PNA) Ng's dispersion was lyophilized using TFD5503, Ilshin Lab, Co. Ltd., and the % practical yield was quantified gravimetrically (eqn (1)). Freeze dried 5-FU–PNA Ng's were screened for FT-IR, ¹H-NMR, and particle size. FT-IR and ¹H-NMR were carried out as per the methodology mentioned in Section 2.3. Particle size, PDI and zeta potential were analysis in distilled water was facilitated by DLS (Nano ZS90, Malvern instruments). The screening tests were carried out at 25 °C and 37 °C.

2.4.2. Morphological analysis of 5-FU–PNA Ng's by transmission electron microscope. Surface morphology of 5-FU–PNA Ng's was analysed using transmission electron microscope (TEM) Jeol, JEM-2100 electron microscope. 1 mg mL⁻¹ 5-FU–PNA Ng's dispersion was placed on formvar carbon coated 200 mesh copper grid (FCF-200-Cu, Electron Microscopy Sciences). 1% w/v phosphotungstic acid was used as a strainer and copper grids were air dried at 20 °C and 40 °C respectively for 1 h. The accelerating voltage was 120 kV.^27,34

2.4.3. In vitro drug release studies. Five mg mL⁻¹ 5-FU–PNA Ng's solution was prepared in PBS pH 5.5, 6.8 and 7.4 respectively.^27,32 From the said solution, 5 mL was transferred to dialysis bag and immersed in 195 mL of PBS of corresponding pH. The drug release studies were carried-out at 37 ± 0.5 °C and 100 rpm. Aliquots were withdrawn at pre-determined time intervals and quantified at 265 nm using UV spectrometer.

2.4.4. Hemolysis analysis. Ng's compatibility with blood was evaluated by hemolysis analysis. Fresh human blood was collected in a CPDA-1 blood bag and was used for the study. 900 μL of blood was transferred to Eppendorf tubes, to which 100 μL of PNA or 5-FU–PNA Ng's in different concentrations (1, 0.5, 1, 2 mg mL⁻¹) was added. The prepared samples were incubated at 37 ± 0.5 °C for 1 h under continuous shaking. Post 1 h, samples were centrifuged at 5000 rpm for 10 min. 200 μL of plasma supernatants was added to 3 mL of 0.01% Na₂CO₃, and absorbance was recorded at 450, 415 and 380 nm respectively, using UV spectrometer. Plasma hemoglobin (Hb) was calculated using following eqn (3). Obtained plasma Hb values were compared with positive control saline and negative control 0.1 N HCl.²⁹

Plasma Hb = {(2 × A₄₅₀) − (A₃₈₀ + A₄₁₅) × 76.26} (3)

2.4.5. Sterilization and sterility studies.
2.4.5.1. Sterilization process. Pre calculated amount of 5-FU–PNA Ng's were sealed in ampoules under nitrogen atmosphere and sterilized using gamma rays at a dose of 15.76 kGy cobalt-60 at (Microtrol, Bangalore, India).
2.4.5.2. Sterility test. 5-FU–PNA Ng's dispersion was prepared by dispersing sterilized sample in water for injection. Prepared Ng's dispersion was inoculated to soybean digestive media and fluid thioglycolate medium and examined for sterility. Sterilized medium was used as negative control and un-sterilized medium was used as positive control. Clouding of medium indicates growth of microorganisms and contamination, while clear/transparent medium indicates un-contaminated.

2.4.6. pH-Dependent cytotoxicity. pH-Dependent cytotoxicity of pure 5-FU and 5-FU–PNA Ng's was evaluated using sulforhodamine B (SRB) assay. 10⁴ T47D and EAC cells were grown in 96 well plate containing 200 μL of Dulbecco's Modified Eagle's medium (DME) respectively. The incubation conditions were kept constant at 37 °C with 5% CO₂ for 24 h. Post 24 h of incubation, the supernatant was separated and replenished with 200 μL of fresh DME/6.8 pH DME buffers (adjusted using dilute HCl) containing 12.5 μg mL⁻¹ of 5-FU and 1 mg mL⁻¹ of PNA and 5-FU–PNA Ng's respectively, and incubated for 4 h under above mentioned conditions.³⁴ After 4 h of incubation, supernatant was removed again, followed by addition of 100 μL of 10% w/v TCA, washed with Millipore water and air-dried. The air-dried plates were stained by using 50 μL per well SRB dye and incubated for 20 min, followed by washing with 1% acetic acid and air dried. 100 μL of 10 mM Tris base was added to each plate and mechanically stirred for 2 min and optical density (OD) was measured at 490 nm using microtiter plate reader (Bio-Rad Laboratories, Inc).

2.4.7. In vivo kinetics. All animals for the study were acquired after protocol approval through Institutional Animal Ethics committee, JSS University, Mysore, India (approval no. 169/2015). The animals were purchased from JSS Medical College, Mysore (receipt no. 40371). The animals authorized for carrying out studies were cared under the supervision of pharmacology department, JSS College of Pharmacy, Mysore, India in compliance with Committee for the Purpose of Control and Supervision on Experiments on Animals (CPCSEA) guidelines.

Twelve albino Wistar rats of either sex weighing 230–260 g were divided into two groups and monitored for one week under quarantine conditions prior to studies. The animals were fed a standard chow diet ad libitum and had free access to water. The two pre-divided groups comprising of six in each, were labelled as standard (5-FU) and test (5-FU–PNA). Intra-peritoneal injection of pure 5-FU and 5-FU–PNA (fixed dose 20 mg kg⁻¹ in water for injection) to the standard (5-FU) and test (5-FU–PNA) group's was facilitated using 1 mL syringe equipped with 26 G needle respectively.³⁵ 0.5 mL blood samples were withdrawn post administration from the jugular vein at pre-determined time intervals. Samples were collected in K3 EDTA tube (CB Plus®, Quantum Biomedicals, India), followed by centrifugation for 10 min at 10 000 rpm at 4 °C. Plasma was separated and stored at −50 °C for further analysis.

Plasma drug concentration was evaluated using ultra-fast liquid chromatography (UFLC), (Shimadzu Prominence LC-20AD), equipped with a 1260 binary pump VL (35 MPa), Prominence SIL-20ACHT auto-sampler and Prominence diode array detector. 5-FU from test plasma was extracted by dispersing 790 μL of acetonitrile, as extracting solvent, to the 200 μL plasma test sample. The resulting samples were vortexed for 10 min and centrifuged for 10 min at 10 000 rpm. Supernatant was filtered using 0.2 μm syringe filter. The obtained solution (10 μL) was analysed using Kinetex® C₁₈ column and pH 6 (20 mM) potassium dihydrogen orthophosphate buffer at a flow rate of 0.8 mL min⁻¹. The retention time (R_t) of 5-FU was found to be 4.9 min.

Pharmacokinetic parameters such as maximum plasma concentration (C_max), half life (t_1/2), area under curve (AUC_0–t), area under curve extrapolated to infinity (AUC_0–∞), area under the first moment curve extrapolated to infinity (AUMC_0–∞), and residence time (MRT) were calculated from plasma-concentration time profile.

2.4.8. In vivo anti-cancer efficacy. All animals for the study were acquired and housed as described in Section 2.4.7. Swiss albino mice of either sex weighing between 25–30 g were used for the study. 10⁶ Ehrlich ascites carcinoma (EAC) cells were injected intra-peritoneal into each mice and monitored for weight gain for 10 days.³⁶ Post ten days animals which have shown drastic gain in body weight were considered as EAC tumour induced mice. 24 mice (6 healthy/normal and 18 EAC induced mice) were taken and categorized into 4 groups (normal, control, standard (5-FU) and test (5-FU–PNA); n = 6). Standard (5-FU) and test (5-FU–PNA) groups were treated with intra-peritoneal injection of pure 5-FU and 5-FU–PNA Ng's respectively. A fixed dose of 20 mg kg⁻¹ 5-FU was used and the entire doses were prepared using sterile water for injection I.P. Four more injections on 2^nd, 5^th, 8^th and 11^th day were administrated after first injection. Un-treated EAC induced mice were considered as a control group and healthy mice were grouped under normal. All the animals were monitored till 15^th day of post first injection and variation in body weight was recorded on daily basis.

2.4.9. Hemanalysis. Blood samples (n = 3) from each group were withdrawn on 15^th day of post first injection, through tail vain. Withdrawn blood was stored in K3 EDTA tube (CB Plus®, Quantum Biomedicals, India). Hemanalysis was performed using haemocytometer (BC-1800, Mindray, Germany).

2.4.10. Histo-pathological study. Fourteenth day post first injection, half of the mice from each group were sacrificed and the heart, liver and kidneys were extracted. The extracted organs were stored in 10% formalin for histo-pathological studies.

2.4.11. Mean life span. All the animals were acquired, housed, EAC induced, grouped and treated as described in Section 2.4.8. Mean life span was calculated by dividing the total number of days animals survived to the total number of animals taken. Each group consists of 6 animals and the study was carried out for 60 days.

3. Results and discussion

3.1. Preparation and evaluation of PNA Ng's

3.1.1. Development of PNA Ng's. The aim of the study was to develop 5-FU loaded pH and thermo-sensitive nanogels that could specifically respond to tumour endosomal pH and extracellular pH, while being dormant to physiological pH at physiological temperature. To achieve the above-mentioned, monomers of NIPAM and AAc were copolymerized via free radical polymerization using poly-functional MBA as a crosslinker. The copolymerization was initiated by anionic initiator KPS at 70 °C. SDS was used as a stabilizer. During free radical polymerization, hydrophilic monomers of NIPAM and AAc in presence of poly-functional crosslinker, react to form a hydrophilic three dimensional network of P(NIPAM) or its copolymers (Scheme 1). The polymerization was carried out at 70 °C for 4 h. To remove un-reacted monomers and SDS, the nanogels reactant suspension was dialyzed for 5 days. Further, dialyzed nanogels suspension was lyophilized and the practical yield of freeze-dried polymer was found to be in the range of 78.83–87.23% (Table 1). Freeze-dried nanogels were characterized using FT-IR and ¹H NMR spectroscopic studies to determine the extent of copolymerization.

Scheme 1 Free radical polymerization of P(NIPAM-co-AAc)/PNA.

3.1.2. Characterization and evaluation of prepared PNA nanogels.
3.1.2.1. FT-IR spectral analysis. FT-IR spectral data of NIPAM, AAc, and prepared nanogels were compared and shown in ESI Fig. S1 and S2.† Spectral analytical data of monomer NIPAM showed functional peaks at 2978 cm⁻¹, 2939 cm⁻¹, 2879 cm⁻¹, 1465 cm⁻¹, 1388 cm⁻¹, 1363 cm⁻¹, 1666 cm⁻¹, and 1543 cm⁻¹. Functional peaks at 2978 cm⁻¹, 2939 cm⁻¹ and 2879 cm⁻¹ representing C–H stretching vibrations of methyl and ethyl groups. Functional peaks at 1465 cm⁻¹, 1388 cm⁻¹ and 1363 cm⁻¹ represent asymmetry and symmetry bending of –CH₃, respectively. Amide I and amide II bands at 1666 cm⁻¹ and 1543 cm⁻¹ were considered to be from C=O stretching and N–H plane bending. Peak at 1249 cm⁻¹ represent C–N stretching vibration of amide. Monomer AAc showed functional peak at 1720 cm⁻¹ representing C=O stretching vibration in carboxylic acid. Further, peaks observed at 3000–4000 cm⁻¹ attributed to stretching vibration of acrylic carboxylic –OH.³⁷

FT-IR spectral data of homo-polymer PNA₀ showed all the functional peaks of monomer NIPAM. Similarly, FT-IR spectral data of all co-polymeric nanogels (PNA₁–PNA_2.5) showed all the functional peaks of NIPAM and AAc without any major shift. The obtained results confirm that polymerization was successful.

3.1.2.2. ¹H NMR spectroscopic studies. ¹H NMR spectral analysis of D₂O swelled homo-polymer PNA₀ and copolymers PNA₂ nanogels were sketched and reproduced in Fig. S3–S5.† The ¹H NMR spectra of PNA₀ showed 4 major peaks at 1.028, 1.464, 1.895 and 3.780. The first 3 represents the following groups present in polymer backbone i.e., –CH₃, –CH₂, –CH, whereas the fourth peak reflects the isopropyl –CH group in NIPAM respectively. As per previous reports, the peak area of the above observed peaks should fall in the ratio of 6 : 2 : 1 : 1. However, as per the result, the measured intensity is in the ratio of 6.07 : 1.88 : 1.17 : 1.00 respectively. These observations are in agreement with previously reported findings.³⁸

Similarly, the prepared copolymers PNA_1.0–PNA_2.5 exhibited similar/infinitesimal difference in peak positions. The difference in peak area is attributed in response to AAc content.

As mentioned earlier % copolymer composition was calculated by comparing peak area at 3.9 ppm, with the total peak area between 0.7 and 2.2 ppm. From the results (Table 1), it is clear that copolymer composition was significantly influenced by feed ratio. However, % composition copolymer PNA_1.5 was found to be insignificant. This occurred because of practical failure or because of selected feed ratios of NIPAM/AAc is very narrow.

FT-IR and ¹H NMR spectral analysis confirmed copolymerization between NIPAM and AAc was successful.

3.1.2.3. pH dependent LCST. LCST of individual polymers in response to change in pH was evaluated by measuring the absorbance at regular temperature intervals. The results obtained were sketched and reproduced in Fig. 1. In order to mimic tumour endosomal and extracellular pH, pH 5.5 and 6.8 PBS were used, whereas pH 7.4 PBS was used to mimic physiological pH.^39–41 We have also studied LCST in 0.9% NaCl saline as physiological blood stream contains about 95–105 mM of Cl⁻ which could affect the release of the drug/LCST.⁴² From the results (Fig. 2) it is evident that pH of the sample solution and polymer composition had significant influence over LCST.

Fig. 1 pH dependent lower critical solution temperature (LCST) of PNA_0–2.5 Ng's.

Fig. 2 pH and temperature dependent hydrodynamic diameter of PNA₂ Ng's.

Effect of sample pH over LCST. The homo-polymer PNA₀ Ng (Fig. 1A) showed sharp LCST phase transition between 30–31.5 °C, without being influenced by pH of sample solution. However for all co-polymeric nanogels (PNA₁–PNA_2.5) (Fig. 1B–E), LCST phase transition was found to be significantly influenced by pH of the sample solution. Increase in PBS pH from 5.5 to 7.4 consequently shifted the LCST to higher temperature. For e.g., LCST of PNA₂ in pH 5.5 and 6.8 was found to be between 38 °C and 55 °C respectively, whereas at pH 7.4 it was found to be higher than 55 °C. This characteristic pH dependent LCST of Ng's is because of presence of carboxylic acid derivative (AAc) in the backbone of Ng's which deprotonate easily at higher pH, consequently resulting in increased hydrophilicity of Ng's.^27,43,44
Effect of polymer composition. With the increase in AAc to NIPAM ratio, LCST phase transition of prepared nanogels shifted to higher temperature. For e.g., LCST of homo-polymer in saline solution was found to be 31 °C, whereas LCST of PNA₁–PNA_2.5 shifted to higher temperatures such as 32 °C, 32 °C, 38 °C and 44 °C respectively. Similar behaviour was observed at different pH 5.5, 6.8 and 7.4 PBS respectively. From the results it was also observed that LCST phase transition of homo-polymer PNA₀ is very sharp whereas, phase transition of copolymers PNA₁–PNA_2.5 were found to be broad and extended. This is because with the increase of AAc monomers within the copolymer network, increased hindrance in polymer chain subsequently leading to dehydration/hydrophobic aggregation and further expands the structural breakdown.³⁸

From the results, we can conclude that at 37 °C PNA₂ Ng's comprising of 97.16% molar of NIPAM and 2.84% molar of AAc, responded sharply to pH 5.5 and were dormant at pH 6.8 and 7.4 respectively, making it a suitable candidate for further evaluations.

3.1.2.4. pH and temperature dependent hydrodynamic diameter. PNA₂ Ng's was studied for pH and temperature dependent hydrodynamic volume phase transition (Fig. 2). Initially, average particle size of prepared Ng's were found to be 68.80, 69.19 and 70.25 nm in pH 5.5, 6.8 and 7.4 respectively at 25 °C. The observed increase in particle size with increasing pH is because of the increase in pH, more and more –COOH deprotonate to anionic –COO–, resulting in internal electrostatic repulsion, and hence increase in particle size. Similarly, with decrease in surrounding pH, intra-chain hydrogen bonding association between NIPAM and AAc leads to shrinking of PNA Ng's at pH 5.5.^45,46 The pH and temperature dependent hydrodynamic volume phase transition were found to be in agreement with pH and temperature dependent LCST studies carried out using UV spectrometer. As discussed earlier, decrease in particle size with increase in temperature is because of Ng's transition from hydrophilic to hydrophobic nature, which in-turn resulted in weakening of hydrogen bonding between water and polymer segments resulting in de-swelling/shrinking.⁴⁶

3.2. 5-FU loaded PNA Ng's

3.2.1. Preparation and evaluation of 5-FU loaded PNA (5-FU–PNA) Ng's. 5-FU loading onto PNA Ng's could be achieved either through (A) drug entrapment within the PNA Ng's matrix or (B) hydrogen bonding of 5-FU with COOH-functional group present on the surface of PNA Ng's (carboxylic –OH is δ−, whereas =O and fluoro present in 5-FU are δ+, interaction between these groups forms a stable hydrogen bond. Similarly, –NH present in 5-FU and =O present in COOH– interacts through hydrogen bonding) (Scheme 2).²⁹ % practical yield of 5-FU PNA Ng's was 79.5%, and % EE of 5-FU loading in PNA₂ Ng's was found to be 34.46%. Average particle size, PDI and zeta potential of 5-FU–PNA Ng's in distilled water at 25 °C and 37 °C was found to be 138.7 and 109.1 nm, 0.262 and 0.243, −21 mV and −14 mV respectively. The observed increase of particle size when compared to placebo Ng's might be because of entrapment of 5-FU within the PNA matrix. FTIR spectral analysis confirmed 5-FU and PNA are compatible and formation of 5-FU–PNA (Fig. S6†).

Scheme 2 Illustration of 5-FU loading onto PNA Ng's.

3.2.2. Morphological analysis of PNA Ng's. Micro-morphology of 5-FU–PNA Ng's were found to be spherical shaped and sized about 110–120 nm (approximate value based on scale) at 20 °C (Fig. 3B). However, upon increasing temperature to 40 °C, 5-FU–PNA Ng's collapsed/shrink and are irregular shaped (Fig. 3C). This phenomenal change could be because of thermally induced dehydration of hydrophobic groups.⁴⁷

Fig. 3 (A) 5-FU drug release from PNMA and PNPA Ng's, micro-morphology of 5-FU PNA Ng's at (B) 20 °C and (C) 40 °C (scale bar 100 nm).

3.2.3. In vitro drug release. In vitro drug release from 5-FU–PNA Ng's was found to be pH and temperature dependent (Fig. 3A). Drug release from 5-FU–PNA Ng's was found to be extended and highly sustained in pH 5.5, 6.8 and 7.4. However, when the drug release was compared with respect to individual pH, drug release in pH 6.8 and 7.4 PBS was quite minimal when compared with pH 5.5. This might be due to the fact that, at pH 5.5 (37 °C), Ng's are near to its LCST temperature that lead to weakening of hydrogen bonding between polymer chains and drug, which eventually lead to more open structure and more drug release from matrix. The observed drug release in pH 6.8 and 7.4 PBS is mostly because of time dependent diffusion from matrix. The initial burst release observed irrespective to pH might be due to residuals of un-entrapped drug on the surface of PNA Ng's. Thus, the % drug release in the above said pH was found to be in the following order pH 5.5 > 6.8 > 7.4 respectively.

3.2.4. Determination of extent of hemolysis. Extent of hemolysis was carried out in-order to evaluate blood and prepared Ng's compatibility. The results were presented in Fig. 4A and B. Results evidence that Ng's did not cause any hemolysis and there will be no risk of hemolysis even on systemic administration. Hence, it can be confirmed that prepared Ng's are compatible with blood components.

Fig. 4 (A) and (B) Hemolysis analysis of PNA₂ Ng's and 5-FU–PNA Ng's, (C) blood concentration time profile of 5-FU in Wistar rats. Inset: magnified blood concentration time profile of 5-FU in Wistar rats ranged from 0–6 h. (n = 6), *p < 0.05 and (D) pH-dependent cytotoxicity of blank (untreated), 5-FU, PNA and 5-FU–PNA.

3.2.5. Sterility studies. 5-FU–PNA Ng's post sterilization did not show any cloudiness in soybean digestive media and fluid thioglycolate medium. Therefore, the sterilization process was found to be effective in sterilizing the product.

3.2.6. pH-dependent cytotoxicity. 5-FU, PNA and 5-FU–PNA Ng's were tested and compared for their pH dependent cytotoxicity on T47D and EAC cancer cells by incubating in pH 7.4 and 6.8 buffers at 37 °C respectively, using SRB assay (Fig. 4D). Cell viability of blank (untreated) was evaluated in the above mentioned pH conditions, for evaluating the effect of incubation pH over cell viability. The results reflect that decreasing incubation pH from 7.4 to 6.8, cell viability of T47D and EAC cancer cells remained unaffected and hence was considered as control group. Placebo PNA Ng's when compared against the control did not show any significant difference in cytotoxicity. Compared to control and placebo PNA Ng's cytotoxicity, 5-FU showed significant decrease in cell viability independent of pH, whereas prepared 5-FU–PNA Ng's exhibited pH dependent cytotoxicity wherein, the decrease was more significant in pH 6.8 when compared to its decrease in pH 7.4. Overall, prepared 5-FU–PNA Ng's was significantly cytotoxic in pH 6.8 when compared to its counterparts. However, this was not the same in case of pH 7.4. This significant pH dependent cytotoxicity of 5-FU–PNA Ng's can be attributed to its pH-dependent LCST.

3.2.7. In vivo kinetics. The blood concentration time profile of 5-FU and 5-FU–PNA Ng's after intra-peritoneal injection was plotted in Fig. 4C. C_max, t_1/2, AUC_0–t, AUC_0–∞, AUMC_0–∞ and MRT_0–∞ for 5-FU and 5-FU–PNA Ng's was calculated after extrapolating blood concentration time profile into non-compartmental model using PK Solver. Obtained pharmacokinetic (PK) data was reproduced in Table 2. From the results it can depicted that 5-FU–PNA Ng's could sustain the drug release, and extend the half-life.

Table 2 Pharmacokinetic parameters of 5-FU and 5-FU PNA Ng's after intra-peritoneal injection^a

Pharmacokinetic parameters	5-FU IP injection	5-FU PNA Ng's
a P < 0.05****; when compared with 5-FU IP injection.
t1/2 (h)	0.35 ± 0.046	5.49 ± 3.83
Cmax (μg mL^−1)	98.19 ± 12.518	76.476 ± 7.50
AUC0–t (μg mL^−1 h^−1)	56.43 ± 13.18	214.19 ± 69.95
AUC0–∞ (μg mL^−1 h^−1)	59.61 ± 13.59	232.40 ± 82.50
AUMC0–∞ (μg mL^−1 h^−2)	40.44 ± 14.14	1488.20 ± 1091.30****
MRT0–∞ (h)	0.66 ± 0.076	5.64 ± 3.20

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3.2.8. In vivo anti-cancer efficacy. Intra-peritoneal injection of EAC tumour cells in mice results in development of liquid tumour, which is well known for their characteristic uncontrollable tumour cell growth in intra-peritoneal cavity and increase in body weight.^48–50 Ascitic fluid is an essential nutritional requirement that supports rapid growth of EAC cells. Rapid increase in ascetic fluid along with growth of tumor meets the nutritional requirement of tumor cells. Increase in body weight observed for tumor bearing mice is attributed to increase in ascetic fluid volume.^51,52 The present study focused to evaluate anti-cancer efficacy in mice by measuring the change in their body weight (Fig. 5A–E).

Fig. 5 Picture of (A) normal, (B) EAC bearing control (untreated), (C) standard (5-FU) and (D) test (5-FU–PNA) treated groups on 15^th day post first injection, (E) anti-cancer efficacy in EAC liquid tumour induced Swiss albino mice. *P < 0.05 (* in red; control vs. standard (5-FU)/test (5-FU–PNA), * in blue; standard (5-FU) vs. test (5-FU–PNA)). *p < 0.05 and (F) mean life span data of normal, control, standard (5-FU) and test (5-FU–PNA) groups.

EAC bearing control group has shown an uncontrollable and significant increase in body weight. Standard (5-FU) and test (5-FU–PNA) group's injected with free 5-FU and 5-FU–PNA exhibited significant reduction in body weight when compared to control group. However when standard (5-FU) and test (5-FU–PNA) groups were compared with each other, test (5-FU–PNA) group treated with 5-FU–PNA Ng's has significantly delayed and controlled the increase in body weight of EAC bearing mice. Therefore, it can be said that the 5-FU–PNA Ng's prepared were highly effective in anti-cancer efficacy in contrast to its counterpart.

3.2.9. Hem-analysis. Hem-analytical data of normal, control, standard (5-FU) and test (5-FU–PNA) groups are reported in Fig. 6. As per the previous reports the normal level of mice RBC, WBC, platelet, and hemoglobin are; RBC: 7 × 10⁶ to 13 × 10⁶ mm⁻³, WBC: 5 × 10³ to 12 × 10³ mm⁻³, platelet: 3 × 10⁵ to 10 × 10⁵ mm⁻³ and hemoglobin: 15.6 to 16.4 g dL⁻¹ respectively.⁵³ From the hem-analytical data, WBC and platelet count were found to be highly affected in EAC bearing control group. However, hem-analytical parameters were marginally improved in both standard (5-FU) and test (5-FU–PNA) groups. From the results it was also found that WBC, platelet and haemoglobin parameters of 5-FU–PNA Ng's treated test (5-FU–PNA) group were found to be marginally improved when compared with 5-FU treated standard group.

Fig. 6 Hem-analytical data of normal, control, standard and test groups. *p < 0.05.

3.2.10. Histo-pathological study. Normal, standard (5-FU) and test (5-FU–PNA) groups were evaluated for histopathology (Fig. 7). The study demonstrated no cardiotoxicity and nephrotoxicity. Histopathology examination of liver from standard (5-FU) revealed severe necrosis, and many cells are hyperchromatic, and anaplastic with enlarged nucleus. Areas of centrilobular cellular infiltration and hyperplasia were also seen with mild cytoplasmic vacuolation. However, liver from test (5-FU–PNA) when compared with standard (5-FU) group revealed minimal hyperchromatic, anaplasia, and cellular infiltration. Many cells were found to have enlarged nuclei and necrosis was not observed. Thus, the test (5-FU–PNA) group exhibited liver with reduced oncopathological lesions like hyperchromasia, anaplasia and nuclear enlargement when compared with standard (5-FU) group.

Fig. 7 Histopathology section heart, liver and kidney from different groups. 200×, scale bar 50 μm.

3.2.11. Mean life span. Mean life span data of normal, control, standard (5-FU) and test (5-FU–PNA) groups has been depicted in Fig. 5F. In EAC bearing control group, initial mortality was observed on 22^nd day and at the end 40^th day all the mice deceased. In contrast, initial mortality in standard (5-FU) group was observed on 24^th day and only one mouse out of 6 survived till 60^th day. % survival for the standard (5-FU) group was found to be 16.66%. Whereas in case of test (5-FU–PNA) group treated with 5-FU–PNA Ng's, first mortality was observed on 36^th day and three mice's have survived till 60^th day. The % survival was found to be 50%. The overall MLS of test group has significantly improved when compared with that of control group (p < 0.05).

4. Conclusion

pH and thermo-sensitive Ng's with the earlier mentioned aim have been successfully developed by cross-linking monomers of NIPAM and AAc. Co-polymer ratio was altered and studied for their effect on pH dependent LCST. Ng's comprising of 97.16% molar of NIPAM and 2.84% molar of AAc were found to be a suitable candidate fitting under pre-mentioned objective and were loaded with 5-FU. In vitro drug release from 5-FU–PNA Ng's was found to be pH and thermo dependent. Pharmacokinetic data depicted that 5-FU PNA Ng's could sustain the drug release, and extend the half-life. In vivo anti-cancer studies has proven that 5-FU–PNA Ng's were highly effective in anti-cancer efficacy when compared with its counterpart free 5-FU, and MST has significantly improved when compared with control group.

Acknowledgements

The author Naga Sravan Kumar Varma V., acknowledges JSS University, Mysuru for financial support through JSS University Junior Research fellowship (JRF) order no. REG/DIR(R)/JSSURF/29(1)/2013-14. Authors are also thankful to (A) Dr H. P. Ramesh, (Retired) Experimental Histopathologist, CSIR—Central Food Technological Research Institute, Mysuru, (B) Dr Hsieh-Chih Tsai, Associate Professor, National Taiwan University of Science and Technology, Taiwan, (C) Mr A. Harikrishna, TEM facility, Centre for Cellular and Molecular Biology, Hyderabad, (D) Mr Rudra Vaghela and Sandeep Kanna, Dept. of Pharmaceutics, JSS College of Pharmacy, Mysuru, (E) Mr Praven, and Mr Preren, Dept. Pharmacology, JSSCP, Mysuru, (F) Dr MVSST. Subbarao and Mr Venu, Dept. of Biochemistry, JSS Medical College, Mysuru, and (G) Dr Dattatri K. Nagesha, Head, Faculty of Life Sciences, JSS University, Mysuru, for their assistance in related to this work. Authors have no conflicts of interest.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra18034e

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