Redox-responsive cystamine conjugated chitin–hyaluronic acid composite nanogels

Abstract

Nanoscale carriers were developed to overcome the challenging barriers for the targeted intracellular delivery of chemotherapeutic agents, in particular within tumors. We demonstrate redox responsive cystamine (Cys) conjugated hyaluronic acid (HA)–chitin (CNG) nanogels for the intracellular delivery of doxorubicin (DOX) within colon cancer cells. Chitin, having a slow degrading property, could make HA to slowly degrade, thus protecting the DOX from a sudden burst release, and HA, being a ligand for the CD44 receptor, are over expressed in colon cancer cells (HT-29). 150–200 nm sized DOX-HA-CNGs and DOX-HA-Cys-CNGs were developed and characterized by DLS, Zeta, TG/DTA, FT-IR, EDAX and rheological techniques. The composite nanogel preparations proved to be safe for intravenous administration because they were non-hemolytic and did not interfere with the coagulation cascade. Flow cytometric and fluorescent microscopic analysis proved the specific internalization of DOX-HA-CNGs within HT-29 cells (CD-44 +ve). MTT assay revealed the superior anti-proliferative activity of DOX-HA-Cys-CNGs in CD-44 +ve HT-29 cells compared to that in CD-44 −ve IEC-6 cells. Thus, HA-Cys-CNGs are proven to be a better carrier for the selective, redox responsive and intracellular delivery of DOX.

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Introduction

In the field of drug delivery, the attention of the researchers has been towards hydrogels because of their innate tunable properties, such as chemical,¹ physical,² thermal³ and pH.⁴ Primarily, hydrogels from natural polymers are used in tissue engineering applications for tissue damage and replacements.^5,6 Their properties have been tuned, which motivated the research on drug delivery applications.⁷ Hydrogels are defined as three-dimensional polymeric networks that have the capability of imbibing water or biological fluids.⁸ Hydrogels containing functional groups, such as hydroxyl, amide and protonated amine groups, have a tendency to absorb and swell under environmental conditions.⁹ These can be linked either by physical or chemical attachments through covalent bonding, hydrogen bonding, van der Waals interactions, or physical entanglements.¹⁰ Although hydrogels are very popular, they have certain drawbacks, which have attracted the attention of the researchers for converting these hydrogels to nanoregimes with more specific and attractive properties.¹¹ Diffusion is the main factor and the biggest drawback in these hydrogel systems, in which signal transduction is significantly lower when compared to nanoregimes.¹² To overcome the abovementioned limitations, hydrogels are converted to nanogels, which have increased the surface to volume ratio. There might be an increase in diffusion, conjugation with antibodies and drug volume. To induce these signals and provide greater functionality, interconnected pores can be introduced in the polymers via a bottom up approach converting hydrogels to nanogels.¹³ These three dimensional polymeric scalar matrices were developed by cross linking the polymers called as “Nanogels”.¹⁴ These nanogels are highly hydrophilic polymers, having an innate swelling property and high drug loading capacity.¹⁵ Because the nanogels were prepared in the hydrophilic environment, the need for surfactant is minimal. Nanogels are synthesized by various approaches, such as the physical interaction of polymers, interfacial sol–gel, polymerizing the monomers, and cross linking pre-processed polymers.¹⁶ These nanogels have greater affinity towards water, and thus the mononuclear phagocyte uptake would be minimal.¹⁷ In general, nanogels with a high surface to volume ratio assist in modifying the surface by chemically conjugating the target moieties for ligand–receptor interaction.^18,19 Overall nanogels demonstrate a potential drug delivery carrier for enhancing the systemic drug delivery of hydrophilic and low molceular weight drugs thereby improving their bioavailability.^20,21

Nanogels have been synthesized using various biopolymers and synthetic polymers, such as chitosan, pullulan,²² cellulose,²³ poly(lactic-co-glycolic) acid (PLGA),²⁴ poly ethylene glycol, and polycaprolactone (PCL).²⁵ One of the most common biopolymer present in the human body is “hyaluronan”.²⁶ Hyaluronan is also known as hyaluronic acid (HA), which is a bioinert, anionic and non-sulfated glucosaminoglycan with an average molecular weight of 140 kDa, similar to that of chitin. The presence of hyaluronan in the human body maintains the integrity of extracellular matrices. HA can also be considered to be a natural moisturizer, which could be due to the presence of hydrophilic molecules in HA.²⁷ A special property of HA is that it acts as a ligand for the CD44 receptor, which is involved in various mechanisms, such as converting the native form of hyaluronan to water, cell migration and cell signaling. The degradation of hyaluronic acid would be faster due to the presence of special enzyme called as “Hyaluronidase”.²⁸ HA is commonly present in tissues, such as the skin, tendons and ligaments, and assists in water retention as well as in the tissue coordination, and the level of HA should be continuously maintained.²⁹ Recently, the native form of HA was used for its anti-aging property.²⁶ The major isoform of CD44 is CD44v6,²⁹ which is over expressed in colon cancer cells, as well as metastatic and non-metastatic tumors.³⁰ An investigation of human breast cancer proved that the connective tissues with a high level of hyaluronic acid are in benign stage. Therefore, the presence and requirement of HA in the tumor region can be considered to detect malignant tumors and also act as marker for detecting the level of cancer growth.³¹ In addition to hyaluronic acid, chitin is also a well known biopolymer used for various applications, such as tissue engineering, drug delivery, and protein delivery. Chitin is similar to the HA connected derivative of glucose consisting of N-acetyl glucosamine, and it is highly cationic in nature due to the presence of an amine group.³² Compared to nanogels, composite nanogels exhibit a different perspective towards therapeutic and diagnostic applications. Composite nanogels can be completely prepared and optimized with simple chemical cross linking between two polymers. The reduced glutathione (GSH) and oxidized form of glutathione (GSSG) mechanism is always maintained to regulate cells, such as cell differentiation, proliferation and apoptosis.³³ The conjugation of cystamine, which contains the disulphide bond in the molecule, is used for drug delivery inside the cells. Cystamine can be easily reduced with the help of a GSH reducing agent inside the cells for the drugs release mechanism.^34,35

In this study, hyaluronic acid (HA) and chitin were used to develop composite nanogels for the glutathione-based controlled delivery of hydrophilic drug doxorubicin. Cystamine dihydrochloride contains a disulphide bond linked between chitin and hyaluronic acid, which acts as a reduced glutathione (GSSG). The oxidized form of glutathione (GSSG) is converted to oxidized glutathione GSH inside the cell to maintain the cellular mechanism, and the release of drugs would occur in a controlled manner. Hyaluronic acid, which acts as a ligand for the CD44 receptor in colon cancer cells, assists in the uptake of these composite nanogels.

Experimental details

(a) Materials

Chitin (degree of acetylation 72.4%, molecular weight 150 kDa) was purchased from Koyo Chemical Co. Ltd., Japan. Hyaluronic acid (MW-140 kDa) was purchased from Qingdao Haitao Biochemical Co. Ltd. China, Cystamine dihydrochloride (MW – 225 g mol⁻¹) was purchased from Alfa Aesar, India. Reduced Glutathione (MW – 307 g mol⁻¹) was acquired from Loba Chemi, India, calcium chloride and methanol were purchased from Qualigens, India, and doxorubicin hydrochloride (Dox) was obtained from Pfizer, India. The HT-29 (colon cancer cells) and IEC-6 (intestinal epithelial) cell lines were purchased from NCCS Pune, India. Media (DMEM) for culturing the cells was purchased from Sigma Aldrich.

(b) Preparation of DOX loaded cystamine conjugated hyaluronic acid/chitin composite nanogels (DOX-HA-Cys-CNGs)

50 mg of HA was dissolved in 5 mL of milliQ water with stirring. 2 mL of the prepared hyaluronic acid solution was slowly added drop wise to an α-chitin solution, which was dissolved in a saturated solution of calcium chloride in methanol and stirred overnight to form the composite gels.³⁶ These composite gels were processed using several washing steps (centrifugation – 20 000 rpm, sonication – 75% amplitude) to form HA–CNG composite nanogels. 5 mg of cystamine dihydrochloride (Cys dissolved in 1 mL of milli-Q water) acts as a cross linker between the chitin and HA. First, 2 mL of HA was incubated with 500 μL of EDC (5 mg mL⁻¹) for 3 h followed by the addition of 500 μL of Cys (5 mg mL⁻¹) and stirred overnight.²⁶ The above prepared complete solutions were added to 0.5% chitin solution (5 mg mL⁻¹) to form a gel like structure. After the gel formation, it was further subjected to sonication and washing steps, resulting in the formation of HA-Cys-CNGs. Anticancer drugs can be loaded into the nanogels either by physical adsorption or diffusion. 400 μg DOX was added to 5 mg of HA-CNGs and HA-Cys-CNGs by incubating DOX with stirring for 24 h, enabling drug adsorption and loading.

(c) Characterization of composite nanogels

The size distribution of the different composite nanogel preparations (DOX-HA-CNGs and DOX-HA-Cys-CNGs) were analyzed by dynamic light scattering (DLS using Zetasizer, Malvern Instruments, USA). The size and surface morphology of the nanogels were further confirmed by SEM (JEOL-JSM – 6490 LA). The stability of composite nanogel suspensions was determined by calculating the surface charge obtained from zeta potential measurements (ZP-Zetasizer, Malvern Instruments, USA). FTIR spectra were recorded to determine the chemical structures of the nanogels based on the bonding vibrations on a Perkin-Elmer spectrum RXI. Thermal degradation studies were performed to determine the thermo-responsive properties of modified composite nanogels at temperatures from 25 °C to 500 °C at a heating rate of 20 °C min⁻¹. The thermal profiles of the control chitin, hyaluronic acid, control composite nanogels, and drug loaded nanogels were obtained by thermogravimetry and differential thermal analysis (TG/DTA) using SII TG/DTA 6200 EXSTAR. Energy Dispersive X-ray (EDAX) (JEOL-JSM – 6490 LA) was used to determine the presence of sulphur in cystamine. The scanning energy for EDAX analysis was 15 keV for an elapsed time of 100 s. In FT Raman spectroscopy, when a laser beam is focused on the sample, the scattered photons exchange energy with the vibrational energy. The vibrative motions in the molecules can be measured. FT-Raman spectroscopy was performed using a 1064 Nd:YAG laser BRUKER RFS 27: stand alone FT-Raman Spectrometer to determine the characteristic peaks of the specific compounds in a spectral range of 50-4000 cm⁻¹ with a resolution of 2 cm⁻¹.

(d) Rheological measurements

Rheological measurements were performed using Rheometer Kinexus Pro, USA, to determine the visco-elastic property and interaction between the constituting polymers. Rheological measurements were performed in dynamic oscillatory mode to determine the elastic and viscous modulus as a function of frequency to calculate the linear visco-elastic region (LVER) of the nanogels. The measurements were performed with cone-plate geometry. For low viscosity nanogels, a 40 mm upper cone and flat bottom plate geometry were used. Initially, the sample was loaded onto the plate and the gap between the nanogel and plate were calculated from the force exerted by its geometry. For low viscosity fluids, like nanogels, the measurements were performed by varying the angular frequency from 0.1 to 10 Hz. In the form of a change in the function of frequency elastic modulus (G′), viscous modulus (G′′) and phase angle (δ) were calculated. Here the samples were studied under a constant temperature, which was maintained using a Peltier apparatus fixed to the instrument.²⁵

(e) Hemolysis assay

The release of iron-containing protein hemoglobin into the plasma causes hemolysis, which can be determined by measuring the number of damaged red blood cells.^38,39 This study was performed to evaluate the hemocompatibility of the drug loaded composite nanogels. Hemolysis can be analyzed based on the soret band-based absorption of free hemoglobin at 415 nm in blood plasma. Different concentrations of composite nanogels, both control and drug loaded nanogels, were taken for the hemolytic assay (250, 500, 750 and 1000 μg mL⁻¹). 100 μL of each of these samples was treated with 1 mL of blood and incubated for 2 h at 37 °C in a shaking incubator chamber. Saline and Triton X100 were used as the positive and negative control for the samples, respectively. The absorbance was spectrometrically measured (BioTek Power Wave XS well plate reader) at 380 nm, 415 nm and 450 nm. The plasma hemoglobin can be calculated using the following equation. This procedure was performed as per the earlier reported literature and hemoglobin was calculated using the following equation. If the sample exceeds the hemolysis value of 5%, then it was considered to be hemolytic.^36,37,40

Amount of plasma Hb (mg dL⁻¹) = {(2A415) − [A380 + A450] × 76.25}

(f) Plasma coagulation study

The interaction of the composite nanogels with the plasma coagulation factors was analyzed by measuring the coagulation time. Platelet poor plasma (PPP) was separated from the peripheral blood by centrifugation at 4000 rpm for 15 min. 50 μL of the prepared concentrations ranging from 250–1000 μg mL⁻¹ were added to 450 μL of platelet poor plasma and kept in a shaking incubator for 30 minutes at 37 °C. After incubation, 50 μL of the above solution was mixed with the pretreated composite nanogels and added to 100 μL of a prothrombin reagent (Diagnostica stago, France), and the time for the plasma to coagulate, i.e., prothrombin time (PT), was measured. In the case of the activated partial thromboplastin time (aPTT) measurements, 50 μL of an aPTT activator (Diagnostica Stago, France) was added to 50 μL of plasma and incubated for 180 s before the addition of 50 μL of 0.025 M CaCl₂. After CaCl₂ treatment, the time taken by the plasma to coagulate was measured as aPTT. The values of PTT and aPTT are the ratio values expressed in seconds. The experiments were performed in triplicate, and saline containing PPP was used as the negative control.^36,37,40

(g) In vitro drug release

The encapsulation efficiency was calculated from a standard curve drawn from the known concentrations of doxorubicin (80, 70, 60, 50, 40, 30, 20, 10, and 5 μg mL⁻¹) to their corresponding OD values. 40 mg of the composite nanogels (HA-CNGs and HA-Cys-CNGs) was loaded with 200 μL of DOX (2 mg mL⁻¹) and incubated for 24 h with stirring. The drug loaded composite nanogels were centrifuged at a rate of 20 000 rpm for 20 min. The supernatant with the un-entrapped DOX was measured using a UV-Visible spectrophotometer (Shimadzu UV-Vis 1700) and the drug concentration was calculated using the linear equation of standard curve.

The in vitro release of DOX from HA-Cys-CNG composite nanogels was studied using the Eppendorf method. 500 μL of DOX-HA-Cys-CNGs dispersed in PBS (pH 7.4) was added to different Eppendorf tubes and the released DOX at different time intervals was quantified by centrifuging each Eppendorf tube, and spectrophotometrically analyzing the supernatant. Two sets of experiments were carried out in the absence and presence of GSH (10 mM).

%DOX released = (Amount of DOX present in the supernatant/Initial amount of DOX entrapped within the nanogel)

(h) Cell culture

HT-29 and IEC-6 were maintained in Dulbecco's modified Eagles medium (DMEM) supplemented with 10% fetal bovine serum (FBS). The cells were incubated in incubator under an atmosphere of 5% CO₂. After reaching confluence, the cells were detached from the flask with Trypsin-EDTA. The cell suspension was centrifuged at 3000 rpm for 3 min, and then resuspended in the growth medium for further study.

(i) Cell uptake studies by fluorescent microscopy

Acid etched cover slips kept in 12 well plates were loaded with HT-29 and IEC-6 cells with a seeding density of 20 000 cells per cover slip and incubated for 24 h to allow the cells to attach to the well. After 24 h incubation, the medium was removed and the wells were carefully washed with PBS buffer. The nanogel preparations at a concentration of 0.25 mg mL⁻¹ were then added along with the media in triplicate to the wells and incubated for a time period of 12 h. After incubation, the media with the sample were removed and the cover slips were processed for fluorescent microscopy. Processing involved washing the cover slips with PBS and fixing the cells in 3.7% para-formaldehyde (PFA) followed by a final PBS wash. The cover slips were air dried and mounted on glass slides using DPX. The slides were then viewed under a fluorescent microscope (Olympus-BX-51) to understand the localization of the doxorubicin-loaded composite nanogels at different depths of dermal penetration.

(j) Cell uptake studies by flow cytometry

Flow cytometry was used to quantify the intra cellular fluorescence of DOX within both normal (IEC-6) and colon cancer (HT-29) cells. The cells were seeded at a density of 50 000 cells per well. After the cell attachment, DOX-HA-CNGs, DOX-HA-Cys-CNGs and DOX-CNGs were added and incubated for 12 hours. After the incubation time, the cells were washed, trypsinised and the fluorescence intensity was later quantified using a flow cytometer with excitation from a 488 nm argon laser using FACS Aria II (Beckton and Dickinson, Sanjose, CA), and emission was measured at 520 nm. A minimum of 10 000 events per sample was analyzed.

(k) Cytotoxicity studies

Cell cytotoxicity (MTT) assay was performed to determine the percentage of viable cells that reduced the tetrazolium component to purple colored formazan crystals. For the cytotoxicity studies, 10 000 cells per well were seeded on a 96 well plate, and after 24 h, the cells were treated with different concentrations of DOX-HA-CNGs and DOX-HA-Cys-CNGs diluted in media. In addition, the same concentrations of DOX-HA-Cys-CNGs were diluted in media containing 10 mM GSH to the cells seeded in the 96 well plates. After 48 h, media was removed and a MTT assay was performed according to the literature.²¹ Using an Elisa micro plate reader, the optical densities were measured at a wavelength of 570 nm.

(l) Statistical analysis

The experiments were carried out in triplicate and values are expressed as mean ± standard deviation (SD). A Student's t-test was used to determine the significance. A probability level of p < 0.05 was considered to be statistically significant.

Results and discussion

HA–chitin composite nanogels were successfully synthesized by an ionic interaction between the carboxyl groups of HA and the hydroxyl group of chitin. Cystamine was conjugated using EDC/NHS conjugation chemistry. Thus, the EDC activated carboxyl group of HA interacts with the amine group at one end of cystamine through an amide bond, and the unreacted amine group undergoes ionic interaction with the hydroxyl group of chitin resulting in a cross linked nanogel system. Fig. 1 represents the hypothetical reaction scheme showing the interaction of these components. Within a cancer cell, GSH will cleave the disulphide bond in the cross-linked cystamine, and thus release the loaded therapeutic agent.³²

Fig. 1 Reaction scheme of composite HA-Cys-CNGs formation showing the interaction between HA, chitin and cystamine.

The anticancer agent, DOX, was loaded onto these composite nanogels followed by an overnight interaction with stirring. During the reaction time, DOX was physically entrapped into the cross linked nanogel mesh, and it was physically adsorbed onto the nanogels. The DOX-HA-Cys-CNGs were further washed thrice to remove the unbound and unreacted components.

Table 1 shows the size distribution of the composite nanogels, both DOX-HA-CNGs and DOX-HA-Cys-CNGs were analyzed using DLS. The synthesis technique resulted in 100 ± 20 nm sized DOX-HA-CNGs and 160 ± 35 nm sized DOX-HA-Cys-CNGs. The conjugation of cystamine increased the size of the composite nanogels, which show the presence of a new moiety, cystamine. The zeta potential of +30 mV and +24 mV for DOX-HA-CNGs and DOX-HA-Cys-CNGs, respectively, indicated the positive surface charge and stability.

Table 1 Size distribution measured by the DLS of DOX-HA-CNGs and DOX-HA-Cys-CNGs composite nanogels

Samples	Size	PDI
DOX-HA-CNGs	100 ± 20 nm	0.293
HA-Cys-CNGs	160 ± 45 nm	0.258
DOX-HA-Cys-CNGs	120 ± 25 nm	0.251
HA-Cys-CNGs in cell culture media	265 ± 35 nm	0.243

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The size of these nanogels increased from 160 nm to 265 nm after dispersing them in the media. This increase in the size could be due to the aggregation caused by the protein binding. However, in the preclinical scenario, these targeted nanogels will be reaching the tumor site within a short time. Therefore, even if there might be some aggregation followed by clearance, these nanosized formulations will enhance the availability of the chemotherapeutic agent at the tumor site.

The SEM images (Fig. 2) of these composite nanogels well correlated with the DLS results, indicating spherical morphologies (but slightly interlinked and agglomerated because these are nanogels) within the 200 nm size range as black spots, which could be due to the water based nanogels and over drying of the sample, along with spherical particles. Cystamine-composite nanogels also showed spherical particles, which could be due to the cross linking of cystamine between hyaluronic acid and chitin to form nanogels. The polydispersive index (PDI) of these composite nanogels was less than 0.3, implying that the nanogels are monodispersed. These composite nanogels can be used as drug delivery vehicles.³²

Fig. 2 SEM images of DOX-HA-CNGs (A) and DOX-HA-Cys-CNGs (B).

Furthermore, to determine the presence of disulphide bond between chitin and hyaluronic acid, FT Raman spectroscopy was used. Fig. 3 indicated the presence of cystamine by showing the presence of a disulphide group in the spectral region around 510 cm⁻¹.^41–44 The peak of the disulphide was very sharp but with low intensity, which is due to its very low concentration in the composite nanogels.

Fig. 3 FT Raman spectra of composite nanogels of HA–chitin (a) and Cys-HA–chitin (b).

FT-IR studies were performed to determine the interaction and bonding of composite nanogels between the polymers, and to confirm the presence of drug loaded in the composite nanogels. The FT-IR spectra (Fig. 4) showed a peak at 1630 cm⁻¹, which corresponds to the strong absorption band of the N–H stretch of chitin. In HA, the strong bands at 3450 cm⁻¹ were assigned to the O–H band, and peaks at 1674 cm⁻¹ and 1536 cm⁻¹ were due to the asymmetric bending (C=O) and symmetric bending (C–O) of the carboxyl group, respectively. The aliphatic stretches (C–H) were observed at 2900 cm⁻¹ and 1450–1500 cm⁻¹, corresponding to the SP orbital stretches of HA. Upon blending of HA with chitin, an amide linkage might be formed between them in the CaCl₂ methanol solvent, which could be indicated by the absorption bands at about 1653.21 cm⁻¹ and 1563.68 cm⁻¹ for the amide I and II bands, respectively.³⁶ The characteristic peaks of doxorubicin are C=C stretch at 1570 cm⁻¹, and =CH stretch at 3610 cm⁻¹, which is overlapped with the hydroxyl group peak around 3600 cm⁻¹. These peaks could be observed in the sample nanogels. The characteristics peaks of the C–S stretch were observed at around 670 cm⁻¹ in the cystamine conjugated composite nanogels, whereas these peaks were not clearly seen in the control composite gels. The C–S stretch peak was weak, thus to further confirm the presence of cystamine in composite nanogels, FT-Raman spectroscopy was performed.⁴⁵

Fig. 4 FT-IR spectra of the composite nanogels α-chitin (a), hyaluronic acid (b), HA–CNGs (c), control cystamine (d), Dox-Cys-HA-CNGs (e).

Fig. 5 shows TGA (left) and DTG (right) curves for the composite nanogels. As seen in Fig. 5(A), from the TGA profile, the first step of the degradation of nanogels at less than 100 °C shows the minimum degradation of 8%, which is due to the presence of water in the compound. The 15% degradation of hyaluronic acid at the same temperature proves the water retention property of HA.⁴⁶ The composite nanogels showed a two-step degradation process at 250 °C and 343 °C. The HA–chitin nanogels were degraded in two steps with a 50% weight loss at 250 °C. The weight losses were considerably slow and gradual degradation occurred at 400 °C when cystamine was conjugated with the HA–chitin nanogels. The weight losses of the Cys-HA–chitin nanogels were comparatively lower than the HA–chitin nanogels at 290 °C to 240 °C. The first step of degradation occurs due to the decomposition of low molecular weight compounds, such as polysaccharides, in the composite nanogels. When the temperature increased to 400 °C, the main backbone structure was degraded with a heavy weight loss.⁴⁷

Fig. 5 TG thermograms (A), DTG (B) for chitin, HA, HA-CNGs, DOX-HA-CNGs and DOX-HA-Cys-CNGs, EDAX (c) plot of HA-Cys-CNG composite nanogel.

Differential thermal gravimetric analysis (DTG) was performed to understand the behavior of cystamine-conjugated composite nanogels with control composite nanogels. As the temperature increases, hydrophilic macromolecules having disordered structure can be easily hydrated. The first order differentiation of TGA was observed by DTG, which explains the mechanism of the decomposition of the material, i.e., whether the process is endothermic or exothermic, where polysaccharides would possess hydration properties depending on the molecular structures in the nanoformulations. Therefore, the control composite nanogels and cystamine conjugated composite nanogels showed a different water retaining capacity, which could be due to the effects of the amorphous nature in the nanoformulations (Fig. 5(B)). Corresponding to TGA, the endothermic and exothermic peaks were interpreted in DTG, which shows the exact degradation point in thermal degradation.^48,49

To determine the presence of cystamine in the composite nanogels, EDAX was performed. This study confirmed the presence of sulphur in the sample with a very small mass percentage of 0.98%, whereas the amounts of cystamine added to the composite nanogels were also similar to the EDAX value in Fig. 5(C). The most dominant compound present was carbon with a mass value of 53%, and sputter coated gold showed a higher value of 45%.

From Fig. 6, the elastic modulus (G′), viscous modulus (G′′) and phase angle (δ) were measured as a function of frequency from 0.1 to 10 Hz. The elastic modulus is independent of frequency until 2 Hz, and there is only slight deviation as a function of frequency. The viscous modulus is completely independent of frequency until 1 Hz; however, the viscous modulus increased and became completely dependent on frequency at more than 1 Hz. This shows that the loss modulus is higher, and when the nanogels start swelling they do not regain their energy, and the swelling nature of nanogels increases until the entire structure is completely deformed and degraded. This can be further confirmed from the phase angle of the nanogels. At the initial frequency, the phase angle is minimal around 15°, and when it reaches 1 Hz, the value of phase angle drastically changes to 50°. Therefore, the stability of the composite nanogels are similar for both the control chitin–HA and Cys-HA–chitin composite nanogels.^50,51

Fig. 6 Rheological measurements of HA-CNG nanogels and HA-Cys-CNGs.

A hemolysis study was conducted for the nanogel systems and the results showed that the hemolytic ratio of the samples varying from 250 μg mL⁻¹ to 1 mg mL⁻¹ was less than 5% of the hemolysis in the RBC cells. These results show that the nanoformulations are safe for IV administration (Fig. 7).

Fig. 7 Hemolysis assay of DOX-HA-CNGs and DOX-HA-Cys-CNG composite nanogels.

To determine the effects of these nanoformulations on plasma coagulation, a PT/aPTT study was performed. Different concentrations of samples were taken from 250–2000 μg mL⁻¹ and treated with platelet poor plasma, and the coagulation time was measured. The normal range of prothrombin time falls in the range of 12–15 s and aPTT values fall in the range of 27–35 s. The PT/aPTT values of both DOX-HA-CNGs and DOX-HA-Cys-CNGs lies in the normal range (Table 2) suggesting that the composite nanogels have no effect on the intrinsic and extrinsic coagulation pathway. Therefore, PT and aPTT measurements proved that the nanoformulations do not interact with the coagulation factor proteins; therefore, they do not cause any interference in the plasma coagulation study.

Table 2 PT and aPTT values of DOX-HA-CNGs and DOX-HA-Cys-CNGs composite nanogels

Dox-HA–chitin			Dox-Cys-HA–chitin
Concentration (μg mL^−1)	PT (sec)	aPTT (sec)	Concentration (μg mL^−1)	PT (sec)	aPTT (sec)
250	15.0	33.4	250	14.5	32.95
500	15.0	31.75	500	14.6	32.0
750	14.9	32.2	750	14.4	32.25
1000	14.8	33.3	1000	14.7	33.45
2000	14.7	31.75	2000	14.4	31.8

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The loading efficiency was calculated with different incubation times with same composite nanogels. Doxorubicin is highly hydrophilic, which prevented the loading of the drug into the polymeric nanoregime matrix. Because the complete system is based on water, the drug easily comes out from both the control and conjugated nanogels. The loading efficiency was not more than 30%. When the incubation time increased from 12 to 24 h, the drug loading was the maximum of 33%. Most of the drug would easily come out when the samples are significantly centrifuged. Similarly, the cystamine conjugated nanogels also showed the same behavior as the control composite nanogels.

In vitro drug release of DOX from the composite nanogels was performed at pH 7.4 in the presence and absence of GSH (Fig. 8). Around 32% of DOX was released from DOX-HA-Cys-CNGs, whereas the presence of 10 mM GSH enhanced the release to 53% within 6 h. Although this could be considered a burst release, GSH had a significant impact on the DOX release. Subsequently, there were very slow releases of DOX for 48 h. For about 2 days, there was a sustained release of DOX from the composite nanogels resulting in a significant difference in the percentage of DOX released due to the presence of GSH. This proves the sensitivity of DOX-HA-Cys-CNGs towards the GSH environment. Cancer cells are reported to have 10 mM GSH, which would cause the enhanced intracellular release of DOX enabling improved cancer cell death.³

Fig. 8 In vitro release in of both drug loaded cystamine conjugated nanogels with 10 mM GSH (Red Square) and without GSH (Green triangle) at the pH of 7.4.

The intracellular localization and the cancer cell targetability of the nanogel preparations were quantitatively demonstrated by fluorescent microscopy (Fig. 9), and qualitatively by flow cytometry (Fig. 10). Because doxorubicin has inherent fluorescence it can emit a red colour that can be used for the analysis.

Fig. 9 Fluorescent microscopy images of HT-29 (A–C) and IEC-6 (D–F) cells. Control cells (A & D), cells treated with DOX-CNGs (B & E), cells treated with DOX-HA-Cys-CNGs (C & F) for 12 h.

Fig. 10 Flow cytometry analysis of the cellular uptake of a nanogel preparation by CD44 +ve cancer cells (HT-29) and CD44 −ve normal cells (IEC-6) after 12 h incubation.

To confirm the localization of the nanogels within the intracellular compartments, the fluorescent intensities of DOX from the nanogel preparations were tracked, analyzed and photographed. Fig. 9 shows fluorescent microscopy images of the HT-29 cells and IEC-6 cells treated with DOX-CNGs, DOX-HA-Cys-CNGs, and the control cells. The enhanced intensity was observed for the DOX within the HT-29 cells treated with DOX-HA-Cys-CNGs compared to that of DOX-CNGs. The enhancement could be due to the fact that HA possesses an affinity towards the CD 44 receptors expressed by the cancer cells. A comparison of CD-44 +ve HT-29 and CD44 −ve IEC-6 cells revealed clear enhanced internalization within the HT-29 cells, proving the target ability of HA towards CD44.²⁹

The cellular uptake of the nanogel preparations were quantitatively analysed by flow cytometry. Fig. 10 presents the histograms obtained from the flow cytometry analysis of HT-29 and IEC-6 cells treated with DOX-CNGs, DOX-HA-CNGs and DOX-HA-Cys-CNGs. Around 31% of HT-29 cells showed the uptake of DOX-CNGs, whereas around 81 to 86% cells had taken up the HA incorporated nanogels. The IEC-6 cells comparatively showed less uptake, where the HA incorporation reduced the uptake of DOX-CNGs from 23% to 6 and 10%. This result supports the fact that HA complexation helps in enhancing the cellular uptake by CD44 +ve cancer cells.

An MTT assay was carried out to determine the toxicity induced by DOX-CNGs, DOX-HA-Cys-CNGs and DOX-HA-Cys-CNGs (with GSH) towards the IEC-6 and HT-29 cell lines. The potency of DOX towards cancer cells is well established when DOX interchelates with DNA and inhibits the biosynthesis of macromolecules as well as the progression of Topoisomerase II, which prevents replication by a DNA binding mechanism. The cell viability profile for HT-29 cells (Fig. 11A) showed significant enhancement in the toxicity induced by DOX-HA-Cys-CNGs and DOX-HA-Cys-CNGs (with GSH) compared to that of the DOX-CNGs to CD44 +ve HT-29 cells. Although these composite nanogels induce toxicity towards CD44 −ve IEC-6 cells (Fig. 11B), there is no statistical significance observed when the groups were compared. Therefore, the study shows that HA assisted the CD-44 mediated uptake of these nanogels, and the intracellular GSH within HT-29 cells might have reduced the disulphide bond releasing more DOX and inducing more toxicity. In addition, in the case of DOX-HA-Cys-CNGs (with GSH), the presence of GSH in the media itself caused –S–S– bond cleavage increasing the amount of DOX and significantly causing enhanced cancer cell death. This did not occur within the CD 44 −ve IEC-6 cells. The cells carry out normal endocytosis of the nanosized moieties and the potent DOX caused the cell death. Therefore, this investigation clearly shows that HA enhanced the targeted delivery of DOX towards CD44 +ve HT-29 compared to that of CD44 −ve IEC-6 cells.

Fig. 11 Cell viability profile of HT-29 (A) and IEC-6 (B) cells treated with DOX loaded composite nanogels for 24 h and analyzed by the MTT assay. Representative hypothetical scheme of the mechanism of toxicity induced in a CD 44 +ve HT-29 cancer cell (C). * and # represent the statistical significance between DOX-CNG and DOX-HA-Cys-CNG and DOX-CNG and DOX-HA-Cys-CNG (with GSH).

Conclusions

In summary, we have developed composite nanogels based on chitin and hyaluronic acid. Cystamine was conjugated with HA–chitin composite nanogels and loaded with the anticancer agent, doxorubicin. These composite nanogel preparations were characterized by DLS, SEM, FTIR, and rheological measurements. The nanogels within 200 nm size range showed redox responsive behavior in the presence of GSH. Flow cytometry quantitative analysis and fluorescent microscopy qualitative analysis proved the selective uptake of DOX-HA-CNGs by CD 44 +ve colon cancer cells (HT-29) compared to that of the CD-44 −ve normal epithelial cells (IEC-6). HA incorporation assisted in CD-44 targeting of these nanogels to HT-29 colon cancer cells, thus inducing redox responsive and enhanced cancer cell death. Therefore, this redox responsive CD-44 specific nanogel system can be used as a better drug delivery agent for the targeted intracellular delivery of therapeutic agents for efficient cancer treatment.

Acknowledgements

The authors acknowledge the Department of Biotechnology (DBT), Government of India for supporting this work under the Nanoscience and Nanotechnology Initiative program (Ref. no. BT/PR10850/NNT/28/127/2008), and for providing financial support. Author S. Maya is thankful to Council of Scientific and Industrial Research (CSIR), India for providing Senior Research Fellowship (SRF Award no.: 9/963 (0012) 2K11-EMR-I).

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Footnote

† Authors contributed equally.

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