pH-Responsive Semi-IPN Nanoparticles Based on 4‑Carboxyphenylboronic Acid-Modified Chitosan for Targeted and Controlled Release of Chemotherapeutic Drug

Abstract

Chitosan nanoparticle-based drug delivery systems incorporating 4-carboxy-phenylboronic acid (PBA) have emerged as a key strategy for enhancing therapeutic efficacy by active targeting of tumor and improving drug loading capacity. Herein, we have developed a PBA-modified chitosan-based (PBA-CS) semi-interpenetrating polymer network (semi-IPN) nanoparticles (NPs) of 4-carboxy-phenylboronic acid-conjugated chitosan-poly­(1-vinylimidazole) (PBA-CS-PVIm) and 4-carboxy-phenylboronic acid-conjugated chitosan-poly­(methacrylic acid) (PBA-CS-PMAA) via free radical polymerization of 1-vinyl imidazole (VIm) and methacrylic acid (MAA), with high drug loading capacity of doxorubicin (Dox). The synthesized semi-IPN NPs were characterized using Fourier transform infrared spectroscopy (ATR-FTIR), transmission electron microscopy (TEM), and thermogravimetric analysis (TGA). TEM analysis revealed that the Dox-loaded semi-IPN NPs were spherical with an average diameter of 33.86 nm for PBA-CS-PVIm and 31.28 nm for PBA-CS-PMAA. Correspondingly, DLS measurements showed high positive surface charges, with zeta potentials of +41.1 and +35.9 mV, respectively. The PBA-CS-PVIm semi-IPN NPs showed a higher loading capacity (LC) and encapsulation efficiency (EE) of 180 ± 6 mg/g and 66 ± 3%, respectively, for Dox at pH 5.5, while the LC and EE of PBA-CS-PMAA semi-IPN NPs are 100 ± 5 mg/g and 60 ± 4%, respectively. The pH-responsive biological macromolecules (PBA-CS, PMAA, and PVIm) effectively enabled sustained drug release, resulting in 78% of Dox being released from PBA-CS-PMAA and 65% from PBA-CS-PVIm over 120 h. The faster release from PBA-CS-PMAA was attributed to electrostatic repulsion between Dox and NPs, while the slower release from PBA-CS-PVIm was due to π–π stacking interactions. The semi-IPN NPs demonstrated excellent biocompatibility in Vero cells, whereas Dox-loaded NPs produced significant cytotoxicity in HeLa cells (80%–95%) when incubated for 48 h. Kinetics studies revealed that the release of Dox follows diffusion and polymer relaxation mechanisms, fitting both the Higuchi and Korsmeyer–Peppas models. These results demonstrate the potential of PBA-CS-based semi-IPN NPs as efficient nanocarriers for chemotherapy.

1. Introduction

Cancer is one of the leading causes of death globally, while chemotherapy has gained prominence for its effectiveness against cancer cells. Conventional chemotherapy suffers from a variety of drawbacks, including low water solubility, effectiveness, lack of specificity, dosage selectivity, quick drug metabolism, multidrug resistance, and susceptibility to degradation in acidic conditions. Among them, doxorubicin (Dox) is one of the most potent anticancer agents, but its clinical use is restricted due to its severe toxicity toward healthy tissues. Dox can cause nephrotoxicity, hepatotoxicity, alopecia, bone marrow suppression, cognitive dysfunction, and, most critically, cardiotoxicity. To reduce these adverse effects while enhancing therapeutic performance, we have explored Dox-loaded nanoparticles as a more controlled and stable delivery strategy. In recent years, nanoparticle-based targeted drug delivery systems have gained significant attention. In particular, interpenetrating polymer networks (IPNs) and semi-IPN nanoparticles have shown great promise for controlled and stimuli-responsive drug release. Semi-IPN and IPN nanoparticles are polymeric systems in which one or more networks are physically interlocked and molecularly entangled without forming covalent bonds between distinct polymer chains, while each polymer preserves its intrinsic covalent architecture. The drug-release behavior of semi-IPN systems can be precisely tuned by adjusting factors such as pH, temperature, and polymer composition. IPNs are especially advantageous because of their biodegradability, biocompatibility, low antigenicity, high swelling capability, improved stability, effective encapsulation of hydrophobic drugs, and potential for tissue-specific delivery.

Polymeric nanocarriers offer flexible platforms for delivering multiple pharmacological agents, aiming to improve therapeutic outcomes and overcome drug resistance in cancer. Synthetic polymers are commonly employed as drug vehicles; however, they suffer from various limitations, including biocompatibility issues and nonbiodegradability. Biopolymers, such as chitosan, the second most abundant biopolymer derived from chitin, offer potential solutions to these challenges. Chitosan has been frequently used in the pharmaceutical field for its biodegradability, biocompatibility, and biochemical activity. The amino and hydroxyl groups on the chitosan offer easy modification with new functional groups. Despite its promise in drug design, chitosan still suffers from challenges such as poor water solubility, low drug loading capacity, limited target specificity, and stability issues. Chitosan nanoparticles (NPs) have been extensively studied as a nanocarrier for chemotherapy. ,

In our previous study, we reported the synthesis of tumor-targeted drug delivery system (DDS), a covalently cross-linked semi-interpenetrating polymer network (Semi-IPN) nanoparticles of chitosan-poly­(methacrylic acid) (CS-PMAA) and chitosan-poly­(1-vinyl imidazole) (CS-PVIm) loaded with doxorubicin (Dox) with superior drug loading capacity as well as encapsulation efficacy. The polyelectrolytes PMAA and PVIm influence nanoparticle (NP) stability through pH-dependent protonation and deprotonation of their functional groups. Since the polyelectrolyte was accessible on the surface of the NPs, they were only protonated at a low pH of 5.0 associated with tumors, resulting in enhanced drug release. However, this system relied on passive targeting via the enhanced permeation and retention (EPR) effect, which is driven by the dysfunctional lymphatic drainage and leaky blood vessels in the tumor microenvironment (TME). Unfortunately, various factors can weaken the EPR effect or restrict the penetration of drug carriers into the tumor region.

To overcome these limitations, 4-carboxyphenlyboronic acid (PBA), a tumor-homing ligand, has been included in NPs-based DDS. It is widely explored in biomedical applications involving the detection of saccharides and other diol compounds, such as sialic acid-binding, , nuclear-targeting, sugar-binding, ATP-responsive, and ROS-responsive. Moreover, PBA offers advantages in cancer cell targeting, such as high affinity and selectivity for sialic acid (SA), as well as being nontoxic, nonimmunogenic, cost-effective, stable, and easily synthesized. Sialic acids (SA) are naturally occurring 9-carbon monosaccharides that are often part of complex glycans of higher animals. The N-acetylneuraminic acid (Neu5Ac) is considered the most important SA, often found at the ends of membrane-bound glycan motifs of various organisms. Abnormal glycosylation is linked with diseases, including cancer. An important indicator of cancer progression is a high level of sialylation. It has been demonstrated that a majority of malignant carcinoma cells exhibit an overexpression of sialic acid groups on their surface. This serves as a potential target site for a boronic acid group, which can interact with the SA residues to form a cyclic boronate ester. PBA-containing materials demonstrated a high drug-loading capacity for chemotherapy drugs like Dox, due to the interaction between PBA and diol groups, along with the coordination between PBA and amines. , At the same time, the high concentration of SA in cancer cells could later excite dissociation and release of the drug-loaded nanocarriers. As a result, these specialized nanocarriers demonstrate enhanced ability to penetrate tumor tissues and deliver anticancer drugs directly to tumor cells due to the precise interactions between ligands and their corresponding receptors.

Since nanocarriers often reach target sites via blood circulation, positively charged carriers, such as chitosan nanoparticles, can attract negatively charged plasma proteins, leading to phagocyte clearance and reduced drug delivery efficiency. Neutral or electronegative carriers face electrostatic repulsion with cell membranes, while electropositive carriers better target endothelial and tumor cells but can accumulate in the liver, causing side effects. Incorporating pH-sensitive groups, such as amino, phosphoric acid, and carboxyl, into carriers can alter their charge properties with respect to environmental pH, improving drug delivery efficiency. For example, pH-responsive carriers can convert to positive charge in tumor environments, enhancing uptake by tumor cells and drug release. Furthermore, pH-responsive polymers can alter the properties of nanodrug carriers to control drug release. Polymers like poly­(methacrylic acid), poly­(aspartic acid), and poly­(vinyl imidazole) become more hydrophilic at lower pH levels, affecting the assembly and disassembly of boron copolymers. The formation of phenylboronic ester bonds in PBA-Dox nanoparticles is responsive to pH, leading to controlled drug release.

Herein, we initially conjugated PBA onto CS using carbodiimide chemistry. Thereafter, pH-responsive PBA-CS-PMAA and PBA-CS-PVIm semi-IPN NPs were produced and considered for the targeted drug delivery of Dox. Our meticulously designed semi-IPN NPs are anticipated to exhibit a remarkable therapeutic effect for several reasons including high drug loading capacity, extended circulation time, effective tumor cell accumulation, and pH-responsive Dox release property. The resulting nanoparticles were evaluated for ATR-FTIR, DLS, and Zeta potential measurements. The nanoparticles were also subjected to morphology examination through transmission electron microscopy (TEM) and other physicochemical characterizations. Furthermore, the loading capacity and encapsulation efficiency of semi-IPN NPs were investigated indirectly. In vitro release behavior of Dox at different pH conditions was monitored via a UV–vis spectrometer. Cytotoxicity of the prepared NPs was conducted to confirm their biocompatibility with healthy cells.

2. Experimental Section

2.1. Materials

Chitosan (DD 81.53% and MW 268 kDa) was extracted from shrimp shell in the laboratory. 4-Carboxyphenylboronic acid (C₇H₇BO₄, MW 165.94g/mol, 99% (w/w)) (PBA), dimethyl sulfoxide (DMSO, 100% (v/v)), N-hydroxysuccinimide (NHS, 99% (w/w)), and 1-ethyl-3­(3-demethylamine propyl) carbodiimide hydrochloride (EDC, 99% (w/w)) were purchased from Sigma-Aldrich, Germany. 1-Vinyl imidazole (VIm 99% (v/v)), methacrylic acid (MAA 99% (v/v), N,N’-methylene bis acrylamide (MBA 99% (w/w)), ammonium persulfate (APS 100% (v/v)) were bought from Sigma-Aldrich, USA. Doxorubicin hydrochloride (Dox) was obtained from Aristopharma, BD. Sodium hydroxide (NaOH), sodium chloride (NaCl), acetic acid (CH₃COOH, 99% (v/v)), ethanol (C₂H₅OH, 99% (v/v)), sodium phosphate dibasic (NaHPO₄), potassium chloride (KCl), and monobasic potassium dihydrogen phosphate (KH₂PO₄) were purchased from Merck, Germany. All experiments were conducted using double-distilled water.

2.2. Synthesis of Chitosan (CS) and 4-Carboxy Phenylboronic Acid Conjugated Chitosan (PBA-CS)

CS was obtained from shrimp shells following the procedure described by Islam et al. The waste shrimp shell was boiled for 1 h and then thoroughly washed multiple times with hot water. The washed shells were oven-dried at 105 °C for 24 h and subsequently crushed in a milling machine. The crushed shells were deproteinized by alkali treatment, mixing with 4% (w/w) NaOH in a 1:16 ratio (w/w) at 70–90 °C for 3 h. The mixture was repeatedly washed until neutralization and dried in an oven at 105 °C for 24 h. The dried samples were demineralized by treatment with 1 N HCl at a ratio of 1:16 (w/w) with constant stirring for 3 h. This was followed by washing with distilled water until neutralization and drying in an oven at 105 °C for 24 h, resulting in the production of chitin. The chitin was then deacetylated by treating with 50% NaOH solution at a ratio of 1:20 (w/w) at 90–100 °C for 4 h. The excess NaOH was removed by washing the product with distilled water until neutralization. Finally, the resulting material was vacuum-dried in an oven to obtain lyophilized chitosan. The degree of deacetylation (DD) of chitosan (CS) was evaluated using ATR-FTIR spectroscopy, while the viscosity-average molecular weight was measured with a viscometer and calculated using the Mark–Houwink equation.

PBA-CS was synthesized by using EDC/NHS as a coupling agent, as shown in Scheme a. Briefly, 0.25% chitosan was dissolved in 1% (v/v) acetic acid solution for 12 h at room temperature. PBA-NHS ester was formed by dissolving PBA (320 mg, 1.9 mM), EDC (740 mg, 3.8 mM), and NHS (440 mg, 3.8 mM) in 50 mL of DMSO and stirring under dark conditions at room temperature for 1 h. Then, PBA-NHS ester solution was slowly added into 250 mL chitosan solution under continuous stirring. The reaction proceeded at 37 °C for 24 h in the dark. The pH of the resultant solution was raised to 10 slowly by using 1 M NaOH for complete precipitation. The resulting product was centrifuged followed by washing and finally lyophilized to obtain dry PBA-CS flakes.

1. Synthesis Route of 4-Carboxyphenylboronic Acid-Conjugated Chitosan (PBA-CS) (a) and Dox-Loaded Semi-Interpenetrating Polymer Network (Semi-IPN) Nanoparticles (b). (Created with BioRender.com).

2.3. Synthesis of PBA-CS-PVIm and PBA-CS-PMAA Semi-IPN NPs

PBA-CS-PVIm and PBA-CS-PMAA semi-IPN nanoparticles were developed through free radical polymerization according to our previous report. Initially, PBA-CS (100 mg) was dissolved in 1% (V/V) acetic acid solution with continuous stirring for 12 h. The solution was filtered using a 0.45 μm syringe filter, and the pH of the resultant solution was raised to 4.8 using 1 M NaOH. The solution was transferred into a round-bottom flask, followed by the addition of 1-VIm and MBA at different ratios (Table ) in a three-neck round-bottom flask equipped with N₂ inlet and condenser with constant stirring for 30 min at 60 °C. The polymerization reaction was started after the addition of 1 mL of 0.183 M APS as an initiator and continued for 1 h at 60 °C under constant stirring and N₂ gas purging. The solution was allowed to cool, and an equal volume of ethanol was added to induce precipitation of nanoparticles. The resultant NPs were purified by centrifugation and decantation, followed by washing with double-distilled water, and finally freeze-dried to yield the PBA-CS-PVIm semi-IPN NPs. PBS-CS-PMAA semi-IPN NPs were prepared following a similar procedure.

1. Monomer Ratios and Average Diameters of the Semi-IPN NPs.

	Composition(wt/wt/wt)
Semi-IPN NPs.	PBA-CS	MAA	VIm	MBA	Particle size (nm)	PDI
PBA-CS-PMAA	100	53	-	15	114.1	0.402
PBA-CS-PMAA	100	79	-	15	147.0	0.317
PBA-CS-PMAA	100	105	-	15	157.1	0.259
PBA-CS-PMAA	100	150	-	15	179.0	0.114
PBA-CS-PMAA	100	210	-	15	195.4	0.160
PBA-CS-PVIm	100	-	53	15	128.6	0.224
PBA-CS-PVIm	100	-	88	15	157.5	0.158
PBA-CS-PVIm	100	-	117	15	183.9	0.211
PBA-CS-PVIm	100	-	175	15	218.4	0.219

2.4. Synthesis of Dox-Loaded PBA-CS-PVIm and PBA-CS-PMAA Semi-IPN NPs

Dox-loaded PBA-CS-PVIm and PBA-CS-PMAA semi-IPN NPs were prepared as shown in Scheme b. In brief, 100 mg of PBA-CS was dissolved in 1% (V/V) acetic acid solution with continuous stirring for 12 h. The solution was filtered using a 0.45 μm syringe filter, and the pH of the resultant solution was raised to 4.8 using 1 M NaOH. Dox was added into the solution at a 10:1 (PBA-CS: Dox) ratio and stirred for 48 h at RT under dark conditions. The solution was moved into a round-bottom flask and polymerized to form Dox-loaded semi-IPN NPs through free radical polymerization as described in Section .

2.5. Determination of Degree of Conjugation

The degree of boronate groups conjugated to CS was determined following the method of Kolawole et al. Briefly, a ninhydrin solution (2% w/v) was prepared by dissolving in DMSO with continuous stirring for 12 h in the dark at RT, as shown in Scheme . Both PBA-CS and CS solutions (0.05–0.25% w/v) were prepared by dissolving in 0.1 M acetic acid and stirring for 12 h under dark conditions at RT. Ninhydrin solution (5 mL) and 4 M sodium acetate (1.25 mL) buffer (pH 5.4 ± 0.2) were mixed with 0.5 mL of prepared polymer solutions. The resultant mixtures were incubated in a water bath at 85 °C for 30 min with mild stirring. The degree of conjugation was determined using a UV–vis spectrophotometer at 569.5 nm (UV-1900i, Shimadzu, Japan). A mixture of ninhydrin and sodium acetate buffer solution (4 M, pH 5.4) (4:1) was used as the blank control. The degree of conjugation was calculated based on eq

$$Degree\; of\; conjugation=\left[\frac{({\delta }_{\mathrm{CS}}-{\delta }_{\mathrm{PBA}-\mathrm{CS}})}{{\delta }_{\mathrm{CS}}}\right]\times 100$$ 1

2. Determination of Degree of Conjugation of 4-Carboxyphenylboronic Acid-Conjugated Chitosan (PBA-CS).

where δ_CS = gradient of the calibration curve of the ninhydrin-chitosan chromophore and δ_{PBA –CS} = gradient of the calibration curve of the ninhydrin-PBA-chitosan chromophore.

2.6. Attenuated Total Reflectance–Fourier Transform Infrared Spectroscopy (ATR-FTIR)

The characteristic functional groups of modified chitosan, unmodified chitosan, and semi-IPN NPs were obtained with an FTIR spectrophotometer (Bruker Alpha II, Platinum-ATR, Germany). Solid samples were scanned at a resolution of 4 cm^–1 within the range of 4000–600 cm^–1. To ensure data accuracy, a total of 128 accumulated scans were performed per sample.

2.7. Particle Size and Zeta Potential (DLS)

The semi-IPN NPs were dispersed into 10 mL of PBS at pH 7.4 and pH 5.0 for particle size and zeta potential analyses, respectively, followed by sonication for 6 h. The suspension was inspected for average particle size and zeta potential utilizing nanoPartica SZ-100 V2 (Horiba, Japan) having diode-pumped frequency-doubled laser (532 nm) in the Semiconductor Technology Research Center, University of Dhaka. The information on size and PDI was collected at a backscattering detection angle of 173°, while the zeta potential was detected at 90°. The zeta potential was measured using electrophoresis theory, based on Smoluchowski’s model. The samples were prediluted at a ratio of 1:100 and run 3 times to ensure reliable data.

2.8. Thermogravimetric Analysis (TGA)

The thermal stability of the as-prepared semi-IPN NPs was analyzed using a PerkinElmer instrument (TGA-8000, USA). The experiments were performed in the temperature range 50–700 °C at 10 ◦C/min for each sample under N₂ atmosphere. The analysis was performed in triplicate.

2.9. Dox Loading Capacity and Encapsulation Efficiency

The Dox loading capacity (LC) and encapsulation efficiency (EE) of the prepared semi-IPN NPs were evaluated by first centrifuging the aqueous medium to isolate the semi-IPN NPs. The amount of free Dox in the supernatant was subtracted from the original concentration to determine the LC. The concentration of free Dox was determined by UV–vis spectroscopy at a wavelength of 496 nm. Loading capacity is defined as the percentage of the total dry mass corresponding to the difference between the initial amount of Doxorubicin (Dox) used for nanoparticle (NP) formulation and the residual Dox remaining following NP separation. To determine the dry mass, an aliquot of the hydrated Dox-loaded NPs was collected and subjected to freeze-drying. The entrapment efficiency and drug loading capacity were determined using eqs and , respectively

$$\mathrm{EE}\%=\frac{Total\; amount\; of\; Dox-Amount\; of\; free\; Dox}{Total\; amount\; of\; Dox}\times 100$$ 2

$$\mathrm{LC}\%=\frac{Total\; amount\; of\; Dox-Amount\; of\; free\; Dox}{Total\; weight\; of\; Dox\; loaded\mathrm{semi}-IPN\; NPs}\times 100$$ 3

2.10. In Vitro Cytotoxic Effect Analysis

The cytotoxic effect of PBA-CS-PMAA and PBA-CS-PVIm semi-IPN NPs on healthy cells (Vero cells line) was examined at the Center for Advanced Research in Sciences, University of Dhaka. The as-prepared NPs were suspended in double-distilled water to prepare the stock solution, and the samples were autoclaved to prevent bacterial contamination. The Vero cells, derived from the kidney of an African green monkey, were cultured in DMEM containing 10% fetal bovine serum, 1% penicillin-streptomycin (1:1), and 0.2% of gentamycin. 48-well plates were used to seed the cells (4.0 × 10⁴/200 μL), which were then incubated at 37 °C in 5% CO₂ incubator (Nuaire, USA). The following day, 50 μL of the autoclaved sample was added to each well. After 48 h of incubation, fresh medium was used to rinse out any insoluble samples. Using a trinocular microscope with a camera (Optika, Italy), the cell cultures were examined for any dead cells. Testing of each sample was performed in duplicate.

The anticancer activity of Dox-loaded PBA-CS-PMAA and PBA-CS-PVIm semi-IPN NPs was evaluated in HeLa cells (human cervical carcinoma cell line) utilizing the same procedure. The cells (human cervical carcinoma cell line) were cultured in DMEM (Dulbecco’s Modified Eagle’s medium) supplemented with 10% (v/v) fetal bovine serum (FBS) and 1% (v/v) penicillin-streptomycin (1:1) and 0.2% gentamycin. Cells (2.0×10⁴/100 μL) were seeded onto a 96-well plate and incubated at 37 °C in 5% CO₂ atmosphere. The next day, 25 μL samples were transferred to each well. The cytotoxicity was examined under an inverted light microscope after 48 h of incubation.

2.11. UV–Vis Absorption and Fluorescence Emission Spectroscopy

The optical properties of the nanoparticles were analyzed using a fluorescence spectrophotometer (Hitachi F-7000, Japan) and a double-beam UV–vis spectrophotometer (UV-1900i, Shimadzu, Japan), across a wavelength range of 200–800 nm, with a scan rate of 200 nm/min and a step size of 2 nm. An ultrapure water solution served as the reference. All measurements were conducted postfiltration. For optimal detection on the spectrometer, the sample containing pure Dox was diluted 10-fold after filtration to ensure they fell within the detection limits.

2.12. Transmission Electron Microscopy (TEM) Analysis

The prepared semi-IPN NPs were imaged to understand particle size and morphology, using Talos F200 × G2 TEM (Czech Republic). For TEM analysis, samples were prepared by suspending a small quantity of nanoparticles in ethanol (2 mg/mL) and sonicating them for 3 h. The suspended NPs were immobilized on copper grids (3.05 mm) by taking 5 μL of suspension, dried at RT, and examined.

2.13. In Vitro Dox Release Analysis

The Dox release profiles of PBA-CS-PVIm-Dox and PBA-CS-PMAA-Dox semi-IPN NPs were evaluated in PBS solution at pH 7.4 and pH 5.0, respectively. In brief, 30 mg of Dox-loaded NPs were suspended in 10 mL of deionized water. Subsequently, the solution was transferred into a dialysis bag with a molecular weight cutoff of 14 kDa (Sigma-Aldrich, USA). The dialysis tube was securely sealed at both ends and immersed in 100 mL of phosphate-buffered saline (PBS). The release medium was maintained at pH 5.0 and 7.4, respectively, and incubated at 37 °C under gentle stirring (100 rpm) by using a magnetic stirrer in the dark. At specific time intervals, 5 mL of the PBS medium was collected, and an equal volume of fresh PBS was added to maintain sink conditions. Dox concentration in the sampled medium was determined using a UV–vis spectrophotometer at an excitation wavelength of 496 nm (correlation coefficient was R² = 0.98 and regression equation of line y = 0.0214x – 0.0009). The cumulative release of doxorubicin from the nanoparticles over time was evaluated and plotted according to eq .

$$Drug\; release\; efficiency(\%)=\left(\frac{Total\; amount\; of\; Dox\; released}{Total\; amount\; of\; Dox\; in\; loaded}\right)\times 100\%$$ 4

2.14. Dox Release Kinetic Studies

Understanding the kinetics of Doxorubicin release is essential for revealing the core mechanisms that control release behavior. To explain the Dox release from the prepared semi-IPN NPs, the in vitro release data was analyzed kinetically. In this context, the Dox release data were fitted to four standard kinetic models for evaluation: zeroth-order equation, first-order equation, Higuchi equation, and Korsmeyer–Peppas equation.

The zeroth-order kinetic model illustrates the constant release of drug from the system, with the release rate being independent of its concentration of the dissolved substance.

-Zeroth-order equation:

$$Q_t=K_{0}t$$ 5

The first-order kinetic model defines the concentration-dependent release rate.

-First-order equation:

$$\log (Q_0-Q_t)=\log Q_0-\raisebox{1ex}{$K_{1}t$}\!\left/ \!\raisebox{-1ex}{$2.303$}\right.$$ 6

According to Higuchi, drug release from an insoluble matrix follows a square root-time-dependent profile governed by Fickian diffusion.

-Higuchi equation:

$$Q_t=K_H{t}^{1/2}$$ 7

Korsmeyer–Peppas model also called power law describes drug release from a polymeric system either through diffusion or swelling.

-Korsmeyer–Peppas kinetic equation:

$$Q_t=\frac{M_i}{M}=K{t}^n$$ 8

Here, Q_t is the Dox release amount at time t, Q₀ is the Dox initial concentration in the dialysis tube, and K₁, K_H, K, and K₀ are the first-order, Higuchi, and Korsmeyer–Peppas model, and zeroth-order release constants, respectively. Additionally, M is the amount of drug at equilibrium, M_i is the amount of drug released at time t, and n is the diffusion exponent. The release kinetics of the drug were analyzed using these equations to identify the best-fit model for each formulation.

2.15. Statistical Analysis

A minimum of three independent trials of the experiments were performed. One-way ANOVA was used to compare group differences, with confidence intervals calculated via Origin software (2018). All data are expressed as mean ± standard deviation, with significance set at P < 0.05.

3. Results and Discussion

3.1. Synthesis and Characterization of Chitosan and Boronated Chitosan Derivatives

The process of extracting CS from shrimp shells consists of three main steps: deproteinization, demineralization, and deacetylation. During deproteinization, proteins and other organic materials are removed through alkali treatment, which breaks the chemical bonds between chitin and the associated proteins or organic compounds. In the demineralization step, minerals (mainly CaCO₃) are removed by using acid treatment, which converts them into water-soluble salts. Finally, in the deacetylation stage, chitin is converted into chitosan by treating it with an alkali to remove acetyl groups. The degree of deacetylation (DD) of chitosan (CS) was evaluated using ATR-FTIR spectroscopy, while the viscosity-average molecular weight was measured with a viscometer and calculated using the Mark–Houwink equation. The DD and the viscosity-average molecular weight were found to be 81.53% and 268 kDa (as shown in Figures S1 and S2), respectively. The viscosity of a 0.25 wt % chitosan solution at 20 °C was measured at 11 mPa·s.

Next, 4-carboxyphenylboronic acid-conjugated chitosan (PBA-CS) was synthesized by attaching phenylboronic acid groups to chitosan using N-hydroxysuccinimide (NHS) and N-(3-(dimethylamino)­propyl)-N-ethylcarbodiimide hydrochloride (EDC) as a pair of coupling reagents. EDC first reacts with carboxylic acid of PBA and produces responsive o-acylisourea intermediates, which then undergo nucleophilic substitution in the presence of a strong nucleophile (i.e., −NH₂ group of CS) as shown in Figure . However, o-acylisourea intermediates are unstable in aqueous solution and react immediately after they are dissolved in water. Since oxygen atoms from water can also act as nucleophiles, they can cleave the intermediate and release isourea, which in turn inactivates EDC. To promote the coupling efficacy, N-hydroxy succinimide (NHS) has been used in conjunction with EDC to form a more stable second intermediate before amination. The NHS creates an active ester, with the carboxylic acid linked to the surface. This intermediary ester is highly hydrophilic and stable and hydrolyzes gradually in the presence of water, providing an advantage for coupling reactions. When amine nucleophiles (CS) are available, the NHS ester rapidly undergoes hydrolysis, promoting the creation of an amide bond with PBA.

1.

Synthesis Mechanism of PBA-conjugated chitosan (PBA-CS).

The conjugation of PBA on chitosan was confirmed by ATR-FTIR spectroscopy. Figure represents the ATR-FTIR spectra of chitosan, PBA, and PBA-CS. The spectrum of chitosan exhibited a typical wide peak at 3010–3710 cm^–1 (N–H and O–H stretching), 2850–2980 cm^–1 (aliphatic C–H stretching), 1640 cm^–1 (−CO stretching), 1320 cm^–1 (N–H bending), 1066 cm^–1 (C–O stretching of 2° alcohol), and 1026 cm^–1(C–O stretching of 1° alcohol). For PBA, peaks at 1660, 1583, and 1486 cm^–1 were caused by CC stretching in the benzene ring, and peaks at 803 and 701 cm^–1 appeared from C–H out-of-plane bending on the meta-substituted benzene. Peaks at 1678, 1420, and 1305 cm^–1 were assigned to CO stretching, C–OH bending, and C–O stretching in the carboxyl group, respectively. The absorption at 1365 cm^–1 was also attributed to B–O stretching. In the spectrum of PBA-CS, the disappearance of CO in the carboxyl group of PBA and the emergence of a new absorption peak at 1640 cm^–1 demonstrate the formation of an amide bond. The successful grafting of PBA onto CS was further recognized by distinctive peaks of CC stretching and C–H bending on the phenyl group at 1564 and 704 cm^–1, while stretching of B–O showed peaks around 1375 cm^–1. The findings are in agreement with previous research, indicating the successful grafting of PBA onto CS. ,

2.

ATR-FTIR spectra of chitosan (CS), 4-carboxyphenylboronic acid (PBA), and 4-carboxyphenylboronic acid conjugated chitosan (PBA-CS).

The PBA content in PBA-CS is represented by the degree of substitution (DS), which indicates the number of PBA groups per nitrogen atom in chitosan. The physical properties and reactive activity of PBA-CS are significantly impacted by the DS of PBA-CS. The ninhydrin color test quantified the DS (Figure S1), by determining the remaining fraction of the amino group present after conjugation. The ninhydrin will react with the remaining primary amine group of PBA-CS, leading to the formation of intermediate amine (2-amino-1,3-indandione), which undergoes a condensation reaction with another molecule of ninhydrin to form diketohydrindylidene-diketohydrindamine (DYDA), also called Ruhemann’s purple. The DYDA is a colored reaction product that shows absorbance at 569.5 nm, as shown in Figure S3. The DS of PBA-CS was found to be 45.3% according to eq .

3.2. Preparation and Characterization of PBA-CS-PVIm and PBA-CS-PMAA Semi-IPN NPs

4-Carboxyphenylboronic acid-conjugated chitosan-based NPs were prepared by free radical polymerization (Figure ). The polymerization process of 1-vinyl imidazole and methacrylic acid is a multistep procedure integral to the synthesis of polymer networks utilizing N,N’-methylene-bis- acrylamide (MBA) as the cross-linker and ammonium persulfate (APS) as the initiator. Initially, APS catalyzes the initiation step by decomposing with the aid of water, yielding sulfate radicals (SO₄•^–) which, in turn, initiates the reaction by attacking the double bonds of the monomers. This generates monomer radicals that subsequently engage with additional monomers of MAA/VIm, driving the propagation phase and facilitating the growth of polymer chains (PMAA and PVIm). Ultimately, termination events happen when the radicals are quenched or undergo combination reactions, forming a stable polymer network.

3.

Proposed structures of (a) PBA-CS-PVIm and (b) PBA-CS-PMAA semi-IPN NPs.

The size and distribution of two semi-IPN NPs (PBA-CS-PVIm and PBA-CS-PMAA) could be tuned by changing the reaction conditions such as the monomer concentration, reaction pH, etc. as shown in Table . As the monomer concentration increases from 53 to 210 of MAA and 53 to 175 of VIm, the hydrodynamic diameter of NPs increases from 114.1 to 195.4 nm of PBA-CS-PMAA and 128.6 to 218.4 nm of PBA-CS-PVIm, respectively, given the reaction temperature, pH, time, and initiator concentration fixed. The DLS analysis denoted that the average diameter of synthesized NPs varied within the range 200 nm depending on each monomer ratio (Figure S4).

The size of the nanoparticles plays an essential role in drug delivery efficiency with smaller nanoparticles exhibiting enhanced cellular uptake and optimized pharmacokinetic distribution. Nanoparticles can enter cells through various mechanisms, including adhesive interactions, endocytosis, micropinocytosis, phagocytosis, and direct diffusion. It has been reported that smaller NPs are more efficiently taken up by cells through diffusion or endocytosis, while larger NPs are more likely to enter cells via phagocytosis. Particles below 100 nm demonstrate up to a 6-fold increase in cellular internalization compared to microscale counterparts, improving therapeutic potential. The desired nanoparticle size was found at a ratio of 100:53:15 (PBA-CS: monomer (MAA/1-VIm): MBA) with an acceptable PDI value. Therefore, the ratio was selected for subsequent analysis. As depicted in Figure a,b, PBA-CS-PVIm and PBA-CS-PMAA NPs exhibited a small hydrodynamic diameter of 128.6 and 114.1 nm, along with a relatively narrow size distribution; their polydispersity index (PDI) values were 0.224 and 0.402, respectively.

4.

Hydrodynamic particle size and zeta potential of PBA-CS-PMAA (a, c) and PBA-CS-PVIm (b, d) semi-IPN NPs.

The zeta potentials of the as-prepared semi-IPN NPs (100:53:15) were measured at pH 5.0 (Figure c,d). The as-prepared PBA-CS-PMAA and PBA-CS-PVIm semi-IPN NPs showed a zeta potential value of +32.2 mV and +37.5 mV, respectively. It was found that the surface charge at or above +30 mV provides greater durability of the NPs. The surface charge property of the semi-IPN NPs can be explained by the combined effect of both polymers: PBA-CS and PMAA/PVIm. In acidic conditions, the amino group of PBA-CS gets protonated (−NH₂ → −NH₃ ⁺), which imparts a positive charge to the surface of the nanoparticle. Similarly, the PVIm chain undergoes protonation below pH 6. Therefore, the high positive surface charge on PBA-CS-PVIm was due to the protonation of both the PBA-CS and PVIm. However, the negative charge of the PMAA chain reduces the overall surface positive charge of PBA-CS for PBA-CS-PMAA semi-IPN NPs.

3.3. Fourier Transform Infrared Spectroscopy (ATR-FTIR)

The ATR-FTIR spectra for VIm, MAA, and the as-synthesized PBA-CS-PMAA and PBA-CS-PVIm NPs are displayed in Figure a. The representative absorption peaks of MAA at 2916 cm^–1, 1690 cm^–1, 1628 cm^–1, and 1422 cm^–1 are ascribed to the C–H stretching vibration, −CO stretching, and −CC double bond stretching, C–H bending vibration, respectively. The formation of PMAA through radical polymerization was confirmed by the loss of −CC group of MAA. The presence of −CO group of PMAA was confirmed by a peak at 1700 cm^–1. Further, the creation of new peaks at 1663 and 1538 cm^–1 associated with the symmetric stretching of COO^– of PMAA and −NH₃ ⁺ of PBA-CS indicates an electrostatic interaction between these two groups leading to the formation of semi-IPN system.

5.

ATR-FTIR spectra of (a) PBA-CS, MAA, PBA-CS-PMAA, I-VIm, and PBA-CS-PVIm and (b) Dox, PBA-CS-PVIm, PBA-CS-PVIm-Dox, PBA-CS-PMAA, and PBA-CS-PMAA-Dox.

The VIm shows characteristic absorption peaks at 1632 cm^–1, 1536 cm^–1, 1300 cm^–1, 1113 cm^–1, 1087 cm^–1, and 912 cm^–1 attributed to −CC stretching, −CN stretching, C–N stretching, ring vibration, in-plane ring C–H bending, and ring deformation, respectively. The intensity of the absorption band of −CC at 1636 cm^–1 for 1-vinylimidazole is reduced after polymerization. The observed wide band at 1545 cm^–1, resulting from the interaction between the amide-II (PBA-CS) and −CN (imidazole ring) bands, is a strong indication of hydrogen bonding occurring between PBA-CS and the cross-linked PVIm. The presence of two new peaks at 1421 cm^–1 (−CH₂ from PBA-CS and PVIm) and 1313 cm^–1 (C–N from PVIm) proved the formation of PVIm.

The mechanism of the PBA-CS-PMAA semi-IPN nanoparticles formation involves the radical polymerization of MAA in the presence of N,N′-methylenebis­(acrylamide) (MBA) as a covalent cross-linker, generating a three-dimensional PMAA network. This covalently cross-linked PMAA framework subsequently interacts electrostatically with PBA-CS chains, as confirmed by IR spectra through the appearance of COO^– and −NH₃ ⁺ associated peaks. Similarly, the PBA-CS-PVIm semi-IPN nanoparticles are formed via polymerization of 1-vinylimidazole to produce a cross-linked PVIm network, which interacts with PBA-CS predominantly through hydrogen bonding. This interaction is supported by the reduced intensity of −OH and −NH stretching bands in the 3400–3600 cm^– 1 region, along with the strengthened CN stretching band at ∼1536 cm^– 1, collectively confirming successful semi-IPN formation.

The ATR-FTIR spectra of Dox-loaded semi-IPN NPs are shown in Figure b. Dox shows multiple peaks, viz., N–H and O–H stretching in the broad range of 3000–3700 cm^–1, C–H stretching at 2830–2990 cm^–1, −CO stretching at 1715 cm^–1, N–H bending vibration mode of a primary amine at 1630 and 1580 cm^–1. The bands at 1422 cm^–1 and 880 cm^–1 are ascribed to the CC stretching and C–H vibration mode of the aromatic ring. Additionally, the peaks at 1276, 1114, and 1068 cm^–1 are ascribed to the stretching vibration of C–O. The spectrum of Dox-loaded semi-IPN NPs displays distinct bands at 1414 and 877 cm^–1, confirming the presence of aromatic rings of Dox. Moreover, the absorption band at 1068 cm^–1 (C–O) is more pronounced. Simultaneously, there is a significant amount of hydrogen bond formation that occurred between the polymer and Dox indicated by a broad absorption peak at 3333 cm^–1 (N–H and O–H stretching). Furthermore, the typical signal of the −B­(O–H)₂ group at 1375 cm^–1 shifted to 1378 cm^–1. These outcomes indicate that Dox was encapsulated into the semi-IPN NPs through a strong coordination bond.

3.4. Electron Microscopy Analysis (TEM)

The morphology and sizes of nanoparticles prepared by polymerization of methacrylic acid (or 1-vinylimidazole) in presence of PBA-CS were investigated using TEM micrographs, as shown in Figure . The as-prepared PBA-CS-PMAA semi-IPN NPs presented highly dispersed NPs with homogeneous morphology and a rather spherical shape (Figure a). The particle size distribution histogram (Figure b) indicates a quite uniform particle size distribution. The NPs are spreading from 17 to 25 nm with a mean diameter of approximately 21.58 nm. In contrast, the TEM image for PBA-CS-PVIm displayed distinct morphological characteristics, showing slight aggregation, likely due to interfacial adhesion within the nanoparticles. The size distribution histogram for PBA-CS-PVIm NPs (Figure d) indicates that the majority of the nanoparticles range from 20 to 27 nm, with an average size of 23.81 nm. These dimensions are consistent with those commonly observed for nanoparticles incorporating chitosan. ,,

6.

TEM images and particle-size distribution histogram of PBA-CS-PMAA (a, b), PBA-CS-PVIm (c, d), PBA-CS-PMAA-Dox (e, f), and PBA-CS-PVIm-Dox (g, h).

The drug-loaded nanoparticles evaluated by TEM revealed an increase in particle size. Dox-loaded PBA-CS-PVIm semi-IPN NPs showed particle diameters in the range of 25 to 43 nm with a mean size of 33.86 nm. Similarly, for PBA-CS-PMAA semi-IPN NPs, the size increased after loading with Dox, ranging from 24 to 40 nm, with a mean size of 31.28 nm. From Figure e,g, it is clear that drug-loaded semi-IPN NPs exhibited darker and larger particle size. Helmi et al. observed a similar morphology in Dox-loaded nanoparticles. This result supports that Dox was loaded into the semi-IPN NPs. It is crucial to recognize that the nanoparticle sizes obtained from TEM micrographs tend to be smaller and show a more limited size distribution compared to those measured by DLS. Notably, the particle size measured from the TEM images (PBA-CS-PMAA = 21.58 nm and PBA-CS-PVIm = 23.81 nm) was smaller than that measured by the DLS (PBA-CS-PMAA = 114.1 and PBA-CS-PVIm = 128.6). This variation is expected because the DLS measures the hydrodynamic particle diameter, which includes the swollen state of the particle in DI water. Given that both PVIm and PMAA are highly flexible and hydrophilic polymers, the semi-IPN is capable of significant swelling in solution, contributing to the larger size observed in DLS measurements.

3.5. Thermogravimetric Analysis (TGA)

The thermal characteristics of the semi-IPN NPs were investigated to determine their thermal stability, which is crucial for defining the processing conditions and technical applications. TGA measures changes in the weight of a substance as a function of time or temperature in a controlled environment. As a result, TGA provides information such as the temperature range at which the sample will achieve a fixed chemical composition and the rate at which oxidation, combustion, dehydration, and other reactions occur. The TGA and corresponding DTG thermogram of PBA-CS-PMAA and PBA-CS-PVIm nanoparticles were characterized between 50 and 700 °C, in the N₂ atmosphere, as depicted in Figure .

7.

TGA and DTG thermograms for (a) PBA-CS-PMAA and (b) PBA-CS-PVIm semi-IPN nanoparticles.

The thermograms of PBA-CS-PMAA NPs showed three stages of weight loss. The initial 10% of weight loss happened at the low-temperature range of 50–150 °C, which can be ascribed to the loss of the water physically adsorbed on the surface of the NPs. The next degradation, which caused a 25% weight loss, occurred between 180 and 300 °C. The depolymerization and degradation of polymer chains occurred due to deacetylation, cleavage of glycosidic linkages, and the elimination of volatile compounds associated with PBA-CS. The final decomposition happened between 300 and 600 °C, leading to a 50% weight loss and a residual mass of 15%, which relates to the loss of the pyranose ring and the disintegration of the remaining carbon in CS, as well as due to the decarboxylation of PMAA. , Similarly, the weight loss curve for PBA-CS-PVIm exhibited only two stages of degradation. In the first event, a weight loss of 15% occurred between 50 and 150 °C, which is attributed to moisture removal. A second thermal event involves the major degradation stage, characterized by 60% weight loss over the heating range of 200–600 °C, resulting in a residual mass of 25%. This may be attributed to the decomposition and degradation of PBA-CS and PVIm chains.

Additionally, the DTG thermograms for PBA-CS-PMAA had three degradation peaks centered at 75, 145, and 380 °C (Figure a). This data showed a slight shift compared to the DTG results for CS-PMAA reported in our previous report, with degradation peaks at 76, 233, and 350 °C. While it can be noted that the introduction of PBA increased the maximum degradation temperature for the third peak from 350 to 380 °C, interestingly, the subsequent peak is lowered to 145 °C from 233 °C. This indicates that PBA-CS varied in its ability to hold water and the strength of its interactions with water, possibly pointing to the formation of unique PBA-CS structures. On the other hand, the PBA-CS-PVIm undergoes two degradation steps at 80 and 240 °C (Figure b). The major decomposition occurs at 240 °C, in contrast to our previous report on CS-PVIm, which showed maximum degradation at 280 °C. Therefore, it can be concluded that the introduction of the PBA conjugate onto chitosan alters the degradation temperature of semi-IPN NPs.

3.6. In Vitro Cytotoxic Effect and Anticancer Activity of NPs

The biocompatibility of the drug carrier was evaluated by incubating the synthesized semi-IPN NPs along with the control sample in the Vero cell lines. The cell viability was calculated under an inverted light microscope by checking for changes in the cell morphology, adherence to the culture medium, and cell density. The microscopic images are shown in Figure . The percentage of cell viability as represented in Table showed that both PBA-CS-PVIm and PBA-CS-PMAA NPs did not exhibit any form of toxicity, as the results matched those of the control experiment, which was made up of the solvent used in the dissolution of the NPs. Above 95% of cell viability was reported for all the NPs, and 100% of cell viability was reported for the control media incubated without adding any solvent or NPs. Chitosan is a biopolymer with known biocompatibility similar to both PVIm and PMAA. The results showed that even the degradation products of NPs do not possess any toxic effect on healthy living cells and can be fabricated for drug delivery applications.

8.

Optical microscope image of Vero cell line treated with (a) control sample, (b) control with solvent, (c) PBA-CS-PVIm, and (d) PBA-CS-PMAA semi-IPN nanoparticles.

2. Cytotoxicity Results of PBA-CS-PVIm and PBA-CS-PMAA NPs.

Semi-IPN Nanoparticles	Survival of Vero cells	Remarks
Control	100%	No cytotoxicity was observed in the Vero cell lines
PBA-CS-PVIm	95%
PBA-CS-PMAA	95%

The anticancer activity of Dox-loaded NPs was investigated in cervical adenocarcinoma cells (HeLa). The viability test was evaluated qualitatively; the cells were incubated for 48 h with PBA-CS-PVIm-Dox, PBA-CS-PMAA-Dox, and free Dox at two different concentrations of 0.5 and 1 mg/mL. The images of the cells after the treatment are shown in Figure . The viability percentage of HeLa cells after incubation with Dox and the Dox-loaded NPs at different concentrations is shown in Table .

9.

Images illustrating HeLa cells viability following exposure to different Dox concentrations. Panels (a–c) show cells treated with 0.5 mg mL^– 1 Dox equivalent to (a) free Dox, (b) PBA-CS-PMAA-Dox NPs, and (c) PBA-CS-PVIm-Dox NPs. Panels (a1–c1) correspond to the same formulations at a higher concentration of 1.0 mg mL^– 1 Dox equivalent. Panel (d) represents the negative control (untreated cells), and panel (d1) represents the positive control (cells exposed to solvent).

3. Cytotoxicity Results of Dox, PBA-CS-PVIm-Dox, and PBA-CS-PMAA-Dox Semi-IPN NPs.

Semi-IPN NPs	Concentration of Dox (mg/mL)	Survival of HeLa cells	Remarks
Control	0	>95%	No cytotoxicity was observed in cancer cell
Dox	0.5	<5%	All samples showed significant cytotoxic effect on cancer cell
PBA-CS-PVIm-Dox	0.5	<20%
PBA-CS-PMAA-Dox	0.5	<10%
Dox	1.0	<5%
PBA-CS-PVIm-Dox	1.0	<10%
PBA-CS-PMAA-Dox	1.0	<5%

The Dox-loaded NPs depicted concentration-dependent anticancer activity, whereby increasing the Dox concentration decreased the cell viability of the HeLa cells. At a lower concentration of 0.5 mg/mL Dox equivalent, the free Dox exhibited a cell viability of less than 5% (Figure a), while PBA-CS-PMAA-Dox and PBA-CS-PVIm-Dox NPs showed a cell survival rate of about 10% and 20%, respectively (Figure b,c). Moreover, at a higher concentration of 1 mg/mL Dox equivalent, the Dox-loaded NPs showed a good anticancer activity, with the highest activity in the case of the PBA-CS-PMAA-Dox, which recorded a cell viability of less than 5% (Figure b1,c1). The results agreed with the release study, as the highest release was in the case of PBA-CS-PMAA-Dox-loaded NPs.

Generally, the as-prepared NPs demonstrated less toxicity at lower concentrations in comparison to free Dox. This could be attributed to the cytotoxic nature of Dox, showing toxicity even on normal cells due to the formation of free radicals. However, the entrapment of Dox within the semi-IPN networks of the NPs created a protective barrier that reduces the direct interactions between the drug and cells. Furthermore, the PBA as a targeting ligand enhanced the NPs homing ability, specifically targeting the overexpressed sialic acid epitopes on cancerous cells. Therefore, we can conclude that both nanocarriers are good candidates for the effective delivery of Dox to cancer cells. The NPs will offer several advantages including slow sustained release and longer retention time because once the PBA forms the ester linkage with the sialic acids, the NPs will exhibit active accumulation at the tumor area and pH-induced drug release at a mildly acidic pH of the tumor, the NPs will swell more, ensuring fast drug release.

3.7. Drug Loading Capacity and Encapsulation Efficiency

The encapsulation and loading efficiencies were estimated by means of the standard formula described earlier. The amount of free drug was determined by assessing the absorbance of the supernatant solution collected after the precipitation of nanoparticles during the synthesis of the drug-loaded systems. To determine the LC and EE, semi-IPN NPs were prepared at two distinct pH values of 4.8 and 5.5. The standard calibration curve in Figure S5, with an R² of 0.98199, was employed to calculate the concentration values required for estimating the LC and EE. The PBA-CS-PVIm semi-IPN NPs showed the highest LC of 180 ± 6 mg/g and EE of 66 ± 3%, respectively, for Dox at pH 5.5, while the LC and EE of PBA-CS-PMAA semi-IPN NPs are 100 ± 5 mg/g and 60 ± 4%, respectively.

In our previous study, we reported that the encapsulation efficiency of CS-PMAA and CS-PVIm at pH 5.5 was 52% and 57%, respectively, and LC of CS-PMAA and CS-PVIm was 48 mg/g and 108 mg/g, respectively. It was assumed that the LC and EE of Dox into chitosan-based semi-IPN NPs significantly increased after conjugation with PBA. The results are presented in Table and in Figures S6 and S7.

4. Encapsulation and Loading Efficiency of Dox Loaded into PBA-CS-PVIm and PBA-CS-PMAA NPs Determined by a UV–Visible Spectrophotometer.

pH	Semi-IPN NPs	LC (mg/g)	EE (%)	Particle size (nm)	PDI	Zeta Potential (mV)
pH 4.8	PBA-CS-PMAA-Dox	60 ± 3	41± 6	130.3	0.379	+34.6
	PBA-CS-PVIm-Dox	76 ± 3.5	46 ± 3	148.3	0.213	+39.2
pH 5.5	PBA-CS-PMAA-Dox	100 ± 5	60 ± 4	139.1	0.410	+35.9
	PBA-CS-PVIm-Dox	180 ± 6	66 ± 3	158.1	0.351	+41.1

The effective loading of Dox into PBA-CS-PMAA semi-IPN NPs is attributed to electrostatic interactions and hydrogen bonding. The PMAA chain, with a pK _a value of 5.5, has both negatively charged −COO^– and neutral −COOH groups at reaction pH, rendering it an anionic polymer capable of electrostatic attraction. The electrostatic interaction occurs among the positively charged amino group of Dox and the negatively charged carboxylic group of PMAA. Due to the abundance of hydroxyl groups in both PBA-CS and PMAA, the Dox molecule forms H-bond with the PBA-CS and PMAA chain, enhancing Dox loading on PBA-CS-PMAA NPs. However, effective loading of Dox onto PBA-CS-PVIm is mainly driven by the π-π stacking interaction between the aromatic ring of Dox and VIm ring. The lone pair of N atom in the imidazole ring of PVIm, and the hydroxyl group and amine group of PBA-CS may form H-bonding with the Dox molecule. Therefore, Dox encapsulation into PBA-CS-PVIm NPs is enhanced through H-bonding. The loading of Dox causes an increase in particle size and zeta potential value due to the cationic nature of Dox. However, it was noticed that by raising the reaction pH from 4.8 to 5.5, both LC and EE improved. This can be ascribed to the change of ionic charges concerning pH. With increasing pH, there is an increasing negative charge density of the PMAA chain, while at the same time, the positive charge density of both PBA-CS and PVIm is reduced. Therefore, the semi-IPN nanoparticles impart less positive charge on the surface, enhancing the drug encapsulation.

It is evident that the nanoparticles exhibited a greater quantity of Dox loading following conjugation, as a result of the additional binding of Dox to PBA. The boron atom in the side chain of PBA-CS is electron-deficient, allowing it to form a strong coordination bond with Dox containing primary amine electron donors. As a result, PBA conjugation with chitosan may be beneficial, as it increases the loading of Dox into nanoparticles and selectively targets cancer cells. Similar enhancements in Dox accumulation following conjugation were observed in previous studies. ,

3.8. UV–Vis Absorbance and Fluorescence Emission Spectroscopy

The UV–vis absorbance and emission spectra of pure Dox, Dox-loaded PBA-CS-PVIm, and PBA-CS-PMAA semi-IPN NPs are shown in Figure . Free Dox was also used at the same concentration as the loaded NPs to compare the spectral features of Dox before and after the loading procedure. Dox is an anthraquinone drug showing UV and fluorescent properties.

10.

(a) UV–visible absorption and (b) fluorescence spectra of Dox, Dox-loaded PBA-CS-PMAA, and PBA-CS-PVIm NPs.

As revealed in Figure a, the absorption spectrum of Dox in PBS solution at pH 7.4 shows bands at 288 and a broad band 480–500 nm, related to the two allowed 1A → 1L_a and 1A → 1L_b π→π* transitions polarized along the short and long axis, respectively. Dox consists of an aglycon and sugar (daunosamine) moieties. At pH 7.4, the daunosamine moieties get protonated, whereas the aglycone part remains neutral. Bands at 252 and 233 nm are assigned to the aglycone moiety, with some contribution from the daunosamine moiety. Consequently, the reduction in peak intensity, broadening, and slight shift of the λ-max from 480 to 490 nm in the absorbance spectra of Dox-loaded PBA-CS-PMAA and PBA-CS-PVIm can be interpreted as a possible indication of the successful interaction of Dox and the NPs. A study by Ting Suet al. reported that the absorbance λ-max showed a red shift after Dox was encapsulated into the nanoparticles, which implied that π–π stacking interaction within the drug-loaded nanoparticles was evoked.

Since the Dox molecule acts as a fluorophore, fluorescence spectroscopy can be used to track its self-association and interaction with polymeric carriers. Fluorescence was excited at 480 nm, corresponding to the absorption maximum of Dox, and the emission spectra were noted over a wavelength range from 500 to 800 nm. Figure b shows that pure Dox has a characteristic emission at 560 nm. Herein, the interaction of Dox molecules with PBA-CS-PVIm and PBA-CS-PMAA NPs was evident from the variation in fluorescence spectra intensity of the supernatant liquid obtained from the dispersed Dox-loaded NPs in PBS solution at pH 7.4. The significant intensity decrease indicated the quenching of fluorescence by energy transfer among π–π interaction overlapped systems.

The higher quenching degree in the PBA-CS-PVIm-Dox NPs likely demonstrated reduced π–π stacking within Dox, and a subsequent stronger π–π interaction between Dox and the semi-IPN NPs indicative of increasing adsorption of Dox onto the PBA-CS-PVIm surface further confirming their high loading capacity (discussed in Section ). Moreover, the fluorescence intensity of the supernatant liquid withdrawn from the PBA-CS-PMAA dispersed solution was significantly higher than that obtained from PBA-CS-PVIm. This can be due to the rapid release of Dox from the PBA-CS-PMAA as a result of its surface charge properties.

The binding sites of Dox onto the PBA-CS-PMAA NPs were predominantly via negatively charged carboxylic acids (COO−). In contrast, the imidazole group was responsible for Dox loading in the PBA-CS-PVIm NPs. Comparatively, PVIm contains imidazole groups that can form H-bonds with water, making it relatively hydrophilic. However, the overall hydrophilicity of PVIm depends on the density and accessibility of these imidazole groups. In contrast, PMAA has carboxylic acid groups, which are extremely hydrophilic and can form robust H-bonds with water. Additionally, these carboxylic acid groups can be deprotonated to form carboxylate anions, further enhancing the hydrophilicity. Due to these highly hydrophilic carboxylic acid groups, PMAA is generally more hydrophilic than PVIm. Consequently, PMAA is likely to wet faster than PVIm, as its carboxylic acid groups provide a stronger interaction with water molecules, explaining the higher fluorescence intensity observed, as it was able to release more Dox over the same period of 6 h compared to the sample with PVIm.

3.9. In Vitro Dox Release Analysis

The Dox release profiles of the as-prepared semi-IPN NPs were evaluated in phosphate-buffered saline (PBS) in physiological barriers (pH 7.4) and cancer cell conditions (pH 5.0) at 37 °C for 120 h. Figure represents the pH-dependent Dox release from PBA-CS-PMAA and PBA-CS-PVIm semi-IPN systems. The Dox release profile of PBA-CS-PVIm semi-IPN NPs showed that only 18% of the loaded Dox was released (at pH 7.4) within 120 h. However, Dox release increased to 65% at pH 5.0, indicating an enhanced Dox release profile in response to acidic stimuli. An initial burst release was observed within the first 6 h, and approximately 9% of the Dox was released at pH 5.0. Then, a controlled release of Dox of up to 65% was monitored during 120 h. However, at pH 7.4, there is a lower burst release of Dox (up to 6%) followed by a controlled release. Conversely, the release of Dox from PBA-CS-PMAA semi-IPN NPs was 25% and 78% at pH 7.4 and 5.0, respectively, within the same 120 h. At pH 5.0, approximately 14% of the drug was rapidly released within the first 6 h, followed by a more gradual and sustained release. Over the course of 120 h, around 78% of the drug was released. These findings indicate that the Dox-loaded semi-IPN nanoparticles exhibit an effective pH-sensitive release behavior. The in vitro drug release at pH 7.4 and 5.0 shows a pH-controlled cumulative release over 120 h. The Dox release from semi-IPN NPs was faster at pH 5.0 than at pH 7.4. The primary factors contributing to the faster release of Dox at pH 5.0 are the higher degree of swelling of the semi-IPN NPs due to repulsive forces between polymer chains, electrostatic repulsion between positively charged amino groups of both Dox molecules and PBA-CS chains, weakening of coordinative interaction among amino group of Dox and electron-deficient boron of PBA, and the enhanced solubility of Dox in acidic media.

11.

In vitro drug release profiles of (a) PBA-CS-PVIm-Dox and (b) PBA-CS-PMAA-Dox semi-IPN NPs in PBS solution at pH 5.0 and 7.4 (at 37 °C).

The pH-responsive swelling behavior of PBA conjugated chitosan nanoparticles can be explained in terms of ionizable groups (Figure ). The interaction between the positively charged PBA-CS chain and the negatively charged PMAA chain forms the PBA-CS-PMAA semi-IPN nanoparticles. These two ionizable groupsthe free amino groups of PBA-CS and the carboxylic acid group of PMAAdisintegrate at various pH levels, which may alter the charge density in the polymer networks and affect the swelling behavior of PBA-CS-PMAA. Under acidic conditions, free amino group of PBA-CS and carboxylic group of PMAA undergo protonation, below the pK _a value: −NH₂ is changed to −NH₃ ⁺ and −COO^– is changed to −COOH. The formation of the ionic groups in the polymeric chains leads to repulsion forces, allowing the nanoparticles to swell in acidic media. Moreover, the increasing electrostatic repulsion between Dox and PBA-CS and the decreasing electrostatic attraction between Dox and PMAA enhanced the release of Dox from PBA-CS-PMAA semi-IPN NPs at pH 5.0. However, as the pH increased, both the positively charged amino groups and the neutral carboxylic acid group are deprotonated. Thus, the stronger electrostatic interaction between PBA-CS and PMAA causes the NPs to shrink, hindering the cumulative release of the Dox molecules through the semi-IPN NPs.

12.

Dox release mechanism from PBA-CS-PVIm and PBA-CS-PMAA semi-IPN NPs.

pH-responsive swelling behavior of PBA-CS-PVIm semi-IPN nanoparticles is due to the presence of an imidazole group of the PVIm chain in addition to the free amino group of the PBA-CS chain. Under acidic stimuli, both the imidazole ring of the PVIm chain and the −NH₂ group of chitosan chain undergo protonation. At pH 5.0, the formation of positively charged ionic groups results in the repulsion of polymer chains, which in turn enhances the swelling of NPs and accelerates the release of Dox. As the pH rises, the PBA-CS and PVIm chains deprotonate, diminishing their repulsive attraction and reducing swelling. As a result, Dox release slows at pH 7.4.

The condition of the coordination bond between amino group of Dox molecule and electron-deficient boron of PBA also directs the extreme slow release and high release of Dox at pH 7.4 and pH 5.0, respectively. The amino group of Dox donates an electron pair to the boron of PBA, forming a strong reversible coordination bond, which remains unaffected at pH 7.4. However, at pH 5.0, the amino group gets protonated, which destroys the coordination bond and thus increases the release percentage of Dox. The comparatively gradual release of Dox during blood circulation at a physical pH of 7.4 helped to minimize side effects caused by early leakage, while the acidic-triggered Dox release improved the adequate dosage for antitumor treatment. Therefore, the in vitro release of Dox from PBA-conjugated chitosan nanoparticles is influenced by pH and shows a sustained release profile with a slight initial burst.

The release behavior of Dox varied significantly among the free drug, chitosan nanoparticles, and semi-IPN nanoparticles. Free Dox released rapidly, reaching almost complete release (∼92.8%) within the first 4 h. Chitosan nanoparticles showed a slower release, with ∼30% released in the first 6 h and a total of ∼45% over 120 h at pH 7.4, indicating some control but still a significant initial burst. In contrast, the semi-IPN nanoparticles exhibited a more controlled and pH-responsive release: PBA-CS-PMAA and PBA-CS-PVIm NPs released only 25% and 18% at pH 7.4, respectively, while under acidic conditions (pH 5.0), release increased to 78% and 65%. The initial burst was also significantly reduced (<15%) compared to chitosan NPs. Moreover, our semi-IPN NP system showed extended Dox release property as compared to other formulations (Table ST-2). These results demonstrate that semi-IPN nanoparticles provide better control over Dox release, minimize premature leakage, and enhance targeted delivery under tumor-like acidic conditions.

3.10. Drug Release Kinetics Studies

To understand the in vitro analysis and predict the drug release mechanism from the semi-IPN NPs system, it is important to analyze the release data by fitting it into various kinetic models. This study considered the zeroth-order, first-order, Higuchi model, and Korsmeyer–Peppas drug release models to mathematically evaluate the release mechanism and kinetic release rate of Dox from PBA-CS-PVIm and PBA-CS-PMAA semi-IPN NPs. Figure depicts the nonlinear fitting data to the kinetic models, and the parameters (n, k, and R²) are presented in Table . As seen from Table , according to correlation values (R²), the release mechanism of Dox from PBA-CS-PVIm and PBA-CS-PMAA NPs at pH 5.0 and 7.4 (37 °C) is well described by the Higuchi and Korsmeyer–Peppas kinetic models. Drug release from NPs has previously been reported to fit more than one model by Alfassamet al.

13.

Evaluation of mathematical kinetic models for Dox released from PBA-CS-PVIm and PBA-CS-PMAA semi-IPN NPs at pH 5.0 and 7.4: (a) zeroth-order, (b) first-order, (c) Higuchi, and (d) Korsmeyer–Peppas models.

5. Results of Mathematical Modeling for Dox Release Kinetics from PBA-CS-PMAA and PBA-CS-PVIm.

Semi-IPN		First order		Zero order		Higuchi		Korsmeyer-Peppas
NPs		R2	K (h–1)	R2	K (mg h–1)	R2	K (mg h–1/2)	R2	n	K (mg h–n)
PBA-CS-PMAA-DOX	pH 5.0	0.51	0.01	0.87	0.69	0.98	8	1	0.57	10.91
	pH 7.4	0.55	0.008	0.86	0.22	0.97	2.5	1	0.57	5
PBA-CS-PVIm-DOX	pH 5.0	0.50	0.008	0.85	0.81	0.98	9.5	1	0.66	4.8
	pH 7.4	0.57	0.007	0.80	0.16	0.97	1.95	1	0.57	0.91

The good fit demonstrated by the Higuchi kinetic model with R² values of 0.97–0.98, equally, shows the Fickian release mechanism of the nanoparticles. The Higuchi model is regularly employed to describe the mechanism of drug release for spherical systems as reported by other studies. This is likely because the amino group in Dox is capable of protonation at a low pH (pK _a = 8.3). Due to the protonation of both Dox and PBA-CS in a moderately acidic environment, the electrostatic repulsion results in diffusion release associated with the Fickian release mechanism. The Higuchi dissolution constant (K) was evaluated as part of this study. The value of K increases with an augment in hydrodynamic particle size. It was found that the K of PBA-CS-PMAA and PBA-CS-PVIm NPs at pH 5.0 is 8 and 9.5, respectively, while at pH 7.4, it was 2.5 and 1.95, respectively.

Since both the kinetic models showed high correlation values, Korsmeyer–Peppas kinetic models were further investigated for other parameters, such as the values of n, which is the diffusion exponent signifying the drug release mechanism. In the case of spherical NPs, values of n ≤ 0.5 indicate Fickian diffusion, 0.5 < n < 1.0 indicate (anomalous) non-Fickian transport characterized by both particle erosion and diffusion mechanism, and n = 1.0 indicates Case II transport (zero order). When n is greater than 1, it indicates the Super Case II transport, and when n is less than 0.5, it is related with drug diffusion through a slightly swollen matrix and a water-filled network mesh.

The values of n for formulated semi-IPN NPs ranged between 0.57 and 0.66, representing a non-Fickian mechanism. The delivery of Dox was influenced by both the diffusion of Dox and the swelling and/or dissolution of the polymer’s network. The release of Dox from the nanoparticles is primarily driven by the pH of the surrounding solution, which acts as a key factor in facilitating Dox diffusion. Additionally, the drug release behavior of PBA-CS-PVIm and PBA-CS-PMAA semi-IPN nanoparticles is affected by the structural responses of the semi-IPN network under varying pH conditions. In our previous study, it was reported that the synthesized polymer chain swelled in an acidic medium and reduced in size at neutral pH, which is due to the protonation and deprotonation of the polymer’s active sites. This property explains the kinetic release mechanism of the PBA-CS-PVIm and PBA-CS-PMAA semi-IPN NPs, which conforms to a non-Fickian release mechanism according to the Korsmeyer–Peppas model. The k value of PBA-CS-PVIm NPs is lower than that of PBA-CS-PMAA NPs at both pH 5.0 and 7.4, demonstrating a slower Dox release profile. The value of k is based on the geometry and structure of the NPs. As a result, the release rate of Dox from PBA-CS-PVIm nanoparticles decreases due to the presence of PVIm on the nanoparticle surface, which hinders the diffusion of Dox between sites before it reaches the PBS medium.

Therefore, the release of Dox from PBA-CS-PVIm and PBA-CS-PMAA semi-IPN NPs is governed by both diffusion and polymer relaxation mechanisms, as evidenced by the good fit to both the Higuchi and Korsmeyer–Peppas models. The Higuchi model suggests that diffusion is a significant mechanism, while the Korsmeyer–Peppas model indicates anomalous (non-Fickian) transport, reflecting a mixed mechanism of diffusion and polymer relaxation. This dual fitting implies that environmental conditions such as pH significantly influence the release mechanism, enhancing drug release at lower pH due to protonation and structural degradation. The structural characteristics of the hydrophilic polymers PMAA and PVIm also improve Dox solubility, facilitating a controlled and sustained release profile suitable for precise drug delivery applications (Figure ).

4. Conclusions

The present study reports the facile synthesis of the PBA-CS-PVIm and PBA-CS-PMAA semi-IPN nanoparticle systems that selectively target cancer cells via receptor-mediated endocytosis. PBA-CS was synthesized by using a carbodiimide-mediated coupling method. The 4-carboxyphenylboronic acid groups attached to the nanoparticles function as cancer-specific targeting ligands, facilitating drug loading and enhancing the therapeutic efficacy of the water-insoluble chemotherapy drug, Dox. The developed Dox-loaded semi-IPN nanoparticles exhibited a homogeneous spherical morphology with narrow size distributions and positive surface charges, indicating good colloidal stability in aqueous media. Furthermore, the nanoparticles demonstrated pH-responsive drug release behavior, with slower release at physiological pH (7.4) and accelerated release under acidic conditions (pH 5.0), characteristic of tumor microenvironments. The effectiveness of the Dox-loaded NPs was further confirmed through cytotoxicity analysis. However, while our in vitro results demonstrate pH-responsive swelling and controlled Dox release, they cannot fully replicate in vivo conditions, including enzymatic degradation, protein interactions, dynamic clearance, and tissue-specific barriers. We also anticipated that despite promising preliminary cytocompatibility and drug release profiles, further in vivo studies, pharmacokinetic evaluation, and long-term safety assessments are required before clinical application. Beyond chemotherapeutic delivery, the semi-IPN nanoparticles with high positive surface charge, pH responsiveness, tunable surface chemistry, and structural stability are suitable for gene delivery, controlled release, and codelivery of drugs, genes, or peptide therapeutics. These features highlight their versatility and broader potential in biomedical applications.

The data are available throughout the manuscript and supporting files.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.5c04836.

• Determination of degree of deacetylation (DD) of chitosan, calculation of viscosity average molecular weight of chitosan, UV–vis spectroscopy of Ruhemann’s purple with probable reaction, calibration curve of ninhydrin-CS and ninhydrin-PBA-CS, hydrodynamic particle size of PBA-CS-PMAA at different ratios of monomers, standard calibration graph of Dox, hydrodynamic particle sizes and zeta potential of Dox-loaded PBA-CS-PMAA and PBA-CS-PVIm, and comparison of the prepared semi-IPN NPs system with other formulations (PDF)

M.A.H.: Writingoriginal draft, software, methodology, investigation, formal analysis, data curation. R.S.: Writingoriginal draft, investigation, formal analysis , conceptualization, data curation. M.T.: Writingreview and editing. H.I.: Writingreview and editing. M.S.R.: Writing – review and editing. M.N.K.: Writingreview and editing, writingoriginal draft, supervision, resources, project administration, funding acquisition, data curation, conceptualization.

The authors declare no competing financial interest.