Cancer is one of the top killers in the world, with an estimated 19.3 million new cases and 10 million deaths reported during 2024 [1]. There have been substantial advancements made in oncological research, but conventional chemotherapy must overcome a series of challenges including non-selective dispersion, fast clearance of the drug, systemic toxicity and the development of multidrug resistance [2]. These limitations have opened the door for the development of new drug delivery systems that could offer targeted, sustained and controlled delivery of an anti-cancer treatment while avoiding unwanted effects.

Controlled drug delivery systems aim to revolutionize the delivery of anti-cancer drugs by being able to extend the therapeutic drug concentrations at the site of the tumor while minimizing systemic exposure to the drug [3]. Hydrogels are one of the various types of systems that could provide controlled release and are rapidly gaining interest in controlled drug delivery systems due to several distinguishing characteristics, such as their composition of (usually) natural materials that contain a high concentration of water, biocompatibility, biodegradability and tailored physico- chemical properties [4]. Hydrogels can be treated as three-dimensional networks of hydrophilic polymers arranged in such a manner so as to absorb and retain a high concentration of water while maintaining their original structure. Their porous nature allows the anti-cancer drugs to be loaded into the hydrogels, while also providing a solid matrix which can facilitate controlled drug release through diffusion, polymer degradation or physical stimulation [5].

Natural polysaccharide polymers such as chitosan and alginate have been thoroughly studied for the production of hydrogels due to their biocompatibility, biodegradability, and minimal immunogenicity [6]. Chitosan, which is produced from the deacetylation of chitin, also has useful properties such as, pH responsiveness, mucoadhesive properties, and antimicrobial characteristics. Drug delivery via chitosan offers advantages over other drug delivery mechanisms, its cationic structure can electrostatically interact with negative biological surfaces (e.g., some tissues and cells), can facilitate increased cellular uptake and improve adhesion to tissue than hydrogel drug delivery via other systems [7]. Alginate, a product of brown seaweed, forms stable hydrogels through ionic cross-linking with divalent cations such as calcium. Chitosan and alginate are sometimes placed into hydrogel systems together; chitosan and alginate hydrogels possess some additive benefits including mechanical properties, improved drug loading, and release characteristics [8].

Hydrogels are excellent drug delivery vehicles, however, their application in cancer therapeutics has been limited by the use of hydrogels for rapidly diffusing drugs, a drug loading capacity of the hydrogel, and maintaining zero-order release kinetics. To address these limitations, researchers have turned to nanoparticulate drug delivery systems in conjunction with hydrogels as a composite prototype combining two technologies while minimizing downsides [9]. Nanoparticles may further encapsulate hydrogels as drug reservoirs as a means of achieved release kinetics in some controlled manner, which could lead to improved efficacy of therapy.

Poly (lactic-co-glycolic acid) (PLGA) nanoparticles are appealing carriers to use as anticancer drug delivery vehicles for advanced cancer therapies, not only because of the established safety with the FDA and biodegradability, but also because of the tunable release characteristics and their capability of preventing degradation of the drug itself (10). PLGA undergoes hydrolytic degradation to lactic and glycolic acids which represent naturally occurring metabolites that can be eliminated from the body safely. PLGA can be made to degrade in the body at various rates or possible profiles via modulation of the lactide to glycolide ratio, molecular weight, or size of the particle [11].

Drug-loaded nanoparticles to hydrogels effectively create multiple release controlled mechanisms whereby drug release will be attributed due to nanoparticle degradation and the properties of the hydrogel.This strategy possesses many benefits including: improved drug stability, increase bioavailability, longer release period, lower dosing frequency, and potential for localized drug delivery [12]. In addition to these advantages, the co-delivery of multiple drugs with various release profiles could also be a potential feasible approach to combination therapy.

Environmental responsiveness is another important aspect of these advanced drug delivery systems. pH responsive systems are also especially relevant in cancer therapy due to the slightly acidic environment of tumors (6.5-6.8) vs physiological pH (7.4) [13]. This difference in pH could be harnessed to specifically release drugs from tumor, which could aid in therapeutic selectivity while minimizing systemic toxicity.

Doxorubicin, an anthracycline antibiotic and one of the most commonly-utilized anticancer agents, has substantial cardiotoxicity and other side effects [14]. The mechanism of action includes intercalating with DNA, inhibiting topoisomerase II, and producing reactive oxygen species that lead to tumor cell death. While the poor clinical utility of doxorubicin stems in part from cardiotoxicity, the amount of doxorubicin that can be administered is restricted by dose-dependent cardiotoxicity in human patients, ultimately leading to a cumulative lifetime dose that limits therapeutic value [15]. Controlled release formulations of doxorubicin could reduce systemic toxicity while maintaining or enhancing anticancer activity.

Doxorubicin delivery from nanoparticle-embedded hydrogel systems exemplifies a multidisciplinary approach involving materials science, nanotechnology and pharmaceutical sciences. There are many important considerations in developing these systems that must take several factors into account including: polymer selection, crosslinking method mechanism, nanoparticles characteristics, drug loading methodology, and release mechanisms [16]. In addition, the thorough evaluation of these systems meaningfully involves an evaluation of physicochemical properties, drug release kinetics, biological efficacy, and safety.

Typical in vitro evaluation models are an important aspect of drug delivery systems development and optimization. In vitro cell culture studies using cancer cell lines can yield pertinent information about cytotoxicity, cellular uptake mechanisms, and therapeutic efficacy. One of the most well-studied cancer cell lines, MCF-7, has been used in some way as an appropriate cancer cell model in evaluating anticancer drug delivery systems as it not only has a strong characterization but it also resembles human breast cancer [17]. The biocompatibility testing is equally as important using normal cell lines to ensure the safety of all developed formulations.

Mathematical modeling of drug release kinetics provides mechanistic information about the release process along with the ability to predict the performance in vivo. Many different types of kinetic models can be used to relate the drug release data with the underlying mechanisms of releasing the drug: zero-order, first-order, Higuchi, Korsmeyer-Peppas, and Hixson-Crowell [18]. The use of the appropriate model is highly dependent on the characteristics of the system and observed drug release behavior.

Historically, an increasingly more important role in pharmaceutical development has been the use of quality by design (QbD) approaches for the empirical product and process understanding of the variables. When applying a QbD approach to nanoparticle-embedded hydrogel systems, the parameters identified and critically assessed would include critical quality attributes, risk assessment, formulation and processing parameter optimization [19]. The advantages to the systematic approach is improved product quality, decreased development time and easier approval pathway for regulatory bodies.

The translation of a laboratory-scale formulations into a clinical application involves consideration of the manufacturing of large-scale batches, assessing regulatory requirements, and designing the clinical trial. Clinical-grade material must be made using good manufacturing practice (GMP). Thorough preclinical studies are essential for safety and efficacy prior to human testing [20]. Combination products develop a complex regulatory reasoning because of their dual device-drug characteristics.

This research will help address current drawbacks to anticancer drug delivery systems by developing and fully evaluating novel nanoparticle-embedded hydrogel systems. The proposed study will investigate the formulation of PLGA nanoparticles containing doxorubicin then embedding in a preformed chitosan-alginate hydrogel matrix. The systems will be characterized for physicochemical properties, drug release characteristics, anticancer efficacy, and biocompatibility for determining their applicability for clinical development.

Materials

Poly (lactic-co-glycolic acid) (PLGA, lactide:glycolide ratio 50:50, molecular weight 30,000-60,000 Da), chitosan (medium molecular weight, 75-85% deacetylated), sodium alginate (medium viscosity), doxorubicin hydrochloride, polyvinyl alcohol (PVA, molecular weight 30,000-70,000 Da), calcium chloride dihydrate, and dialysis tubing (molecular weight cutoff 12,000-14,000 Da) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Dichloromethane (DCM), acetone, and phosphate- buffered saline (PBS) were obtained from Fisher Scientific (Pittsburgh, PA, USA). MCF-7 breast cancer cell line and NIH-3T3 fibroblast cell line were procured from American Type Culture Collection (ATCC, Manassas, VA, USA). Dulbecco's Modified Eagle Medium (DMEM), fetal bovine serum (FBS), penicillin- streptomycin, and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) were purchased from Gibco (Thermo Fisher Scientific, Waltham, MA, USA). All other chemicals were of analytical grade and used without further purification.

Preparation of PLGA Nanoparticles

Doxorubicin-loaded PLGA nanoparticles were prepared using a modified double emulsion (W/O/W) solvent evaporation technique [21]. Briefly, 10 mg of doxorubicin hydrochloride was dissolved in 0.5 mL of deionized water to form the primary aqueous phase (W1). This solution was added to 2 mL of DCM containing 100 mg of PLGA and emulsified using probe sonication (Branson Ultrasonics, Danbury, CT, USA) at 40% amplitude for 60 seconds in an ice bath to form the primary emulsion (W1/O).

The primary emulsion was then added dropwise to 20 mL of 1% (w/v) PVA solution under continuous magnetic stirring at 500 rpm to form the secondary emulsion (W1/O/W2). The resulting emulsion was stirred at room temperature for 4 hours to allow complete solvent evaporation. The formed nanoparticles were collected by centrifugation at 15,000 rpm for 30 minutes at 4°C, washed three times with deionized water, and lyophilized using a freeze dryer (Labconco, Kansas City, MO, USA) for 48 hours.

Characterization of PLGA Nanoparticles

Particle Size and Zeta Potential

The hydrodynamic diameter, polydispersity index (PDI), and zeta potential of PLGA nanoparticles were determined using dynamic light scattering (DLS) with a Zetasizer Nano ZS (Malvern Instruments, Worcestershire, UK). Samples were diluted 1:100 with deionized water and measured at 25°C. Each measurement was performed in triplicate.

Morphological Analysis

The surface morphology of lyophilized nanoparticles was examined using scanning electron microscopy (SEM). Samples were mounted on aluminum stubs, sputter- coated with gold using an ion sputter coater (Hummer 6.2, Anatech USA, Union City, CA, USA), and observed under a SEM (JEOL JSM-6510LV, Tokyo, Japan) at an accelerating voltage of 10 kV.

Drug Encapsulation Efficiency

The encapsulation efficiency (EE) and drug loading (DL) were determined by dissolving 5 mg of lyophilized nanoparticles in 1 mL of DCM, followed by extraction with 5 mL of phosphate buffer (pH 7.4). The aqueous phase was analyzed for doxorubicin content using UV-Vis spectrophotometry (Shimadzu UV-1800, Kyoto, Japan) at 485 nm. The EE and DL were calculated using the following equations:

EE (%) = (Amount of drug encapsulated / Total amount of drug added) × 100 DL (%) = (Amount of drug encapsulated / Weight of nanoparticles) × 100

Preparation of Nanoparticle-Embedded Hydrogels

Chitosan solution (2% w/v) was prepared by dissolving chitosan in 1% (v/v) acetic acid solution under magnetic stirring overnight. Sodium alginate solution (2% w/v) was prepared by dispersing alginate in deionized water and stirring until complete dissolution. Doxorubicin-loaded PLGA nanoparticles (20 mg) were dispersed in 5 mL of chitosan solution using bath sonication for 15 minutes.

The nanoparticle-chitosan dispersion was then mixed with an equal volume of alginate solution under gentle stirring. Calcium chloride solution (2% w/v) was added dropwise as a crosslinking agent while maintaining continuous stirring. The mixture was allowed to gel for 30 minutes at room temperature. Control hydrogels without nanoparticles were prepared following the same procedure.

Characterization of Hydrogel Systems

Swelling Studies

Swelling behavior was evaluated by immersing pre-weighed hydrogel samples in PBS (pH 7.4) and buffer solutions at pH 5.5 and 6.8 at 37°C. At predetermined time intervals, samples were removed, surface water was gently blotted with filter paper, and the swollen weight was recorded. The degree of swelling was calculated using:

Degree of swelling (%) = [(Ws - Wd) / Wd] × 100

where Ws is the swollen weight and Wd is the dry weight.

Gel Fraction and Crosslinking Density

The gel fraction was determined by immersing hydrogel samples in excess PBS at 37°C for 72 hours. The samples were then lyophilized and weighed. Gel fraction was calculated as:

Gel fraction (%) = (Wf / Wi) × 100

where Wf is the final dry weight and Wi is the initial dry weight.

Mechanical Properties

The compressive strength and elastic modulus of hydrogels were measured using a universal testing machine (Instron 5567, Norwood, MA, USA) with a 1 kN load cell. Cylindrical samples (10 mm diameter, 5 mm height) were compressed at a crosshead speed of 1 mm/min until 80% strain. The elastic modulus was calculated from the linear region of the stress-strain curve.

In Vitro Drug Release Studies

Drug release studies were conducted using the dialysis bag method. Hydrogel samples (equivalent to 1 mg doxorubicin) were placed in dialysis bags and immersed in 50 mL of PBS (pH 7.4, 6.8, and 5.5) maintained at 37°C with continuous stirring at 100 rpm. At predetermined time intervals (1, 2, 4, 8, 12, 24, 48, 72, 96, 120, 144, and 168 hours), 3 mL aliquots were withdrawn and replaced with fresh medium. Doxorubicin concentration was determined by UV-Vis spectrophotometry at 485 nm. Cumulative drug release was calculated and release kinetics were analyzed using various mathematical models including zero-order, first-order, Higuchi, and Korsmeyer- Peppas models.

Cell Culture Studies

Cell Viability Assay

MCF-7 breast cancer cells and NIH-3T3 fibroblast cells were cultured in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C in a 5% CO2 atmosphere. Cell viability was assessed using the MTT assay. Cells were seeded in 96-well plates at a density of 1 × 10? cells per well and incubated for 24 hours. Various concentrations of free doxorubicin, blank nanoparticles, blank hydrogels, and drug-loaded hydrogel systems were added and incubated for 48 hours. MTT solution (5 mg/mL) was added and incubated for 4 hours. The formazan crystals were dissolved in DMSO, and absorbance was measured at 570 nm using a microplate reader (BioTek ELx800, Winooski, VT, USA).

Cellular Uptake Studies

Cellular uptake of doxorubicin was evaluated using fluorescence microscopy. MCF-7 cells were seeded on coverslips in 6-well plates and treated with free doxorubicin or drug-loaded hydrogel extract for 4 hours. Cells were fixed with 4% paraformaldehyde, nuclei were stained with DAPI, and cellular uptake was observed using a fluorescence microscope (Olympus IX71, Tokyo, Japan).

Statistical Analysis

All experiments were performed in triplicate, and results are expressed as mean ± standard deviation. Statistical analysis was performed using GraphPad Prism 8.0 software. One-way ANOVA followed by Tukey's post-hoc test was used for multiple comparisons. A p-value < 0.05 was considered statistically significant.

Characterization of PLGA Nanoparticles

The physical characteristics of doxorubicin-loaded PLGA nanoparticles are summarized in Table 1. The nanoparticles exhibited a mean hydrodynamic diameter of 185.3 ± 12.4 nm with a narrow size distribution (PDI = 0.198 ± 0.028), indicating uniform particle formation. The zeta potential was -18.7 ± 2.3 mV, suggesting moderate colloidal stability. SEM analysis revealed spherical particles with smooth surfaces and minimal aggregation.

Table 1: Physicochemical Characteristics of PLGA Nanoparticles.

Nanoparticle-embedded hydrogel systems have emerged as an exciting advancement in controlled drug delivery systems, in particular for cancer therapeutics where real- time regulation of drug release is necessary. This research clearly illustrates the formulation and assessment of PLGA nanoparticle embedded chitosan-alginate hydrogels for slow release of doxorubicin which resolves many of the challenges currently presented by traditional means of chemotherapy.

The PLGA nanoparticles were formulated using the double emulsion solvent evaporation method. This technique successfully produced nanoparticles with optimal properties for drug delivery market. The average diameter of the nanoparticles was 185.3 nm, which is in the ideal size range for EPR (enhanced permeability and retention; [22], allowing preferential accumulation in tumor tissues as a result of deliberately leaky vasculature. The ideal size range also allows the nanoparticles to be taken up by cells and avoids rapid clearance by the reticuloendothelial system (RES). The polydispersity index (PDI) of the nanoparticles (0.198) reflects that the nanoparticles are similar in size- less variability means that the drug release kinetics will be more consistent with reproducible therapeutic outcomes.

The moderate negativity of the zeta potential (-18.7 mv), while providing enough colloidal stability, avoids excessive surface charge that could interfere with drug release through protein adsorption and removal from circulation. The encapsulation efficiency (78.2%) compares favourably to, or surpasses, many frequencies identified within the literature for encapsulation of hydrophilic drugs within PLGA nanoparticles, thus validating the use of the double emulsion method for loading doxorubicin [23]. The loading of doxorubicin (7.1%) is within rational reasonable limits considering the drug efficacy needed compared to nanoparticle stability.

To conclude, this research successfully incorporated nanoparticles into chitosan- alginate hydrogels systems and did not significantly change the properties of the hydrogels together with The reduction in swelling potential and mechanical properties of hydrogel systems due to nanoparticle incorporation could be explained by physical interactions between the nanoparticles and the polymer chains leading to the formation of additional crosslinking sites, and decreased free space available for water uptake [24] . But both were small and did not affect the system.

The pH-responsive swelling behaviour of the hydrogel systems is particularly relevant for cancer therapeutic applications as the increased swelling under physiological pH (7.4) versus acidic conditions mimics that of what could occur in a tumouor microenvironment, and the slightly acidic pH (6.5 - 6.8) could modulate drug release rates [25]. This pH-responsive swelling behaviour provides an additional layer of control over drug release from the delivery system that could allow for tumour selective drug release.

The drug release studies were sustained for 168 hours compared to a release that could be shown for the mentioned hydrogels; this is superior to conventional formulations that show burst drug release from a few hours. Drug release perpetuates pH-dependently, with faster release occurring at higher pH values. This release pattern and aforementioned swelling pattern is also explainable by the ionization state of chitosan - under physiological pH, chitosan amino groups will be primarily deprotonated and allow for relaxation of polymer chains and increased drug diffusion [26]. A release pattern with slower drug release in acidic condition is desirable to target tumours while decreasing systemic exposure.

Using mathematical models to conduct kinetic analysis has provided insight into the drug release mechanisms. The high fit to the Higuchi model (R² > 0.97) suggested drug release was mainly due to diffusion in the hydrogel matrix, following Fick's law of diffusion.

Desirable diffusion-controlled release allows for sustained drug delivery with predictable kinetics [27]. Analysis of the release data using Korsmeyer-Peppas with n- values ranging from 0.421-0.456 indicated we were observing anomalous transport, which shows that drug release is affected by both diffusion and polymer relaxation. This dual-release mechanism provides further control of the release kinetics and explains the sustained release profile we observed.

The increase in cytotoxicity from the drug incorporated in the nanoparticle-embedded hydrogel system to MCF-7 cells based on the IC?? value being significantly lower than that of free doxorubicin means that therapeutic efficacy was improved. This improvement could be due to sustained exposure to the drug, better cellular uptake from nanoparticle-mediated delivery, and possible protection of the drug from degradation while in the delivery system [28]. The controlled release profile of the hydrogels gave a sustained release of the drug and maintained the therapeutic concentrations for longer periods of time this is important for drugs such as doxorubicin where potency is concentration dependent.

The improvement in the biocompatibility profile based on decreased cytotoxicity to normal NIH-3T3 fibroblasts represents a substantial improvement over free doxorubicin. This improvement suggests that the controlled release system could possibly reduce systemic toxicity while maintaining or improving anticancer efficacy. The sustained release profile of the hydrogel means that lower peak concentrations could be achieved while maintaining therapeutic levels; this could translate to lower side effects of the drug on clinical applications [29].

The blank nanoparticles and blank hydrogels showed very low levels of cytotoxicity, indicating that the carrier materials were biocompatible. For downstream clinical translation the biocompatibility of the delivery system itself is important, as its safety will need to be demonstrated.The clinical feasibility of this system benefits from our use of FDA approved polymers (PLGA) and materials recognized as safe (GRAS) (chitosan, alginate).

While our cellular uptake studies were not exhaustive or definitive, they imply an increased intracellular presence of doxorubicin using the nanoparticle embedded hydrogel system. This finding might be a result of improved membrane interaction due to the chitosan constituent which is known for its mucoadhesive and cell- penetrating characteristics [30]. Furthermore, the sustained release from the hydrogel matrix may be an important means of maintaining a constant SAP850 driving force for cellular uptake, and delivering consistent therapeutic dosing over an extended time frame.

Mechanistically, the release of drug from our system involves multiple sequential processes: i.e. initial swelling of the hydrogel polymer and drug diffusion from the nanoparticles to the hydrogel matrix and continuous diffusion from the hydrogel network to the surrounding culture media. Furthermore, the PLGA nanoparticles allow the gradual degradation of the nanoparticles with most hydrogel components contributing to a sustained release effect. This is an example of a multi-level control system to achieve sustained and localized drug delivery. For example, localized delivery systems with incorporated hydrogel options, could be injected or surgically implanted directly at the tumor site, providing a means of achieving high local concentrations with reduced systemic exposure. This approach is particularly relevant for tumors that are surgically accessible, and for adjuvant therapy administered following tumor resection. High sustained release profiles could reduce treatment burdens for patients leading potentially improved quality of life.

Nevertheless, it is imperative to acknowledge the limits and challenges.The in vitro nature of this study will require further evaluation to animal models, which can show biocompatibility, pharmacokinetics, and therapeutic efficacy in vivo. All aspects of the stability of the formulation when deteriorated in physiological conditions, the occurrence or not of an immunogenic response, and the long-term safety profile need to be properly addressed for example, the webinar manufacturability, scalability, and cost of production for clinical alleviation.

Future avenues for research could include investigation into optimizing formulation parameters for target release profile for various types of cancer and their regime. Similarly, targeting ligands could enhance tumor selectivity. Another avenue for exacerbating cancer therapy could be to initiate studies into combination drug delivery where dosage is a discrete pharmacokinetic profile; alternatively, the last intervention directs whether drug delivery is appropriate for tumors or tumor are not suitable encouraging girth in the first place in the acting drug delivery initiated.

The regulatory pathway for combination products will have implications given that they are a hybrid of device-drug. The regulatory pathway will be required to demonstrate, in a relevant animal model with relevant preclinical studies, a biocompatibility study, pharmacokinetics study, and efficacy study. The manufacturing process must utilize scalable and reproducible methods to comply with good manufacturing practices (GMP) for the clinical production potential.

This study has clearly elucidated the generation and full assessment of a new nanoparticle-embedded hydrogel for controlled anticancer drug delivery. The PLGA nanoparticles had optimal attributes, with mean diameter of 185.3 nm and encapsulation efficiency of 78.2%. The chitosan-alginate hydrogel matrix provided controlled drug release over 168 hours and pH-mediated behavior conducive to targeting tumor microenvironments.

The viable system has been shown to have greater anticancer efficacy against MCF-7 breast cancer cells with an IC?? of 2.34 μg/mL when compared to free doxorubicin (4.67 μg/mL), and biocompatibility improved against normal cells with controlled drug release consistent with the Higuchi kinetics model (square root of time), which is diffusion-controlled release kinetics relevant for sustained therapy.

Promising work thus far is leveraging the combined hypernanoparticle and hydrogel technology for delivering localized cancer therapy for clinical amelioration. The hybergenic system can overcome many of the challenges associated with conventional chemotherapy drug delivery such as rapid systemic clearance of the drug, systemic toxicity, and indistinct tumor selectivity. Future studies to be initiated should consider in vivo studies and optimizing targeting of the platform system for individual clinical cancer applications.

1. Sung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, et al. Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 CA Cancer J Clin. 2021; 71: 209-249.
2. Senapati S, Mahanta AK, Kumar S, Maiti Controlled drug delivery vehicles for cancer treatment and their performance. Signal Transduct Target Ther. 2018; 3: 7.
3. Mitchell MJ, Billingsley MM, Haley RM, Wechsler ME, Peppas NA, Langer Engineering precision nanoparticles for drug delivery. Nat Rev Drug Discov. 2021; 20: 101-124.
4. Li J, Mooney DJ. Designing hydrogels for controlled drug delivery. Nat Rev Mater. 2016; 1: 16071.
5. Qiu Y, Park K. Environment-sensitive hydrogels for drug delivery. Adv Drug Deliv Rev. 2001; 53: 321-339.
6. Peppas NA, Bures P, Leobandung W, Ichikawa H. Hydrogels in pharmaceutical formulations. Eur J Pharm Biopharm. 2000; 50: 27-46.
7. Dash M, Chiellini F, Ottenbrite RM, Chiellini E. Chitosan—A versatile semi- synthetic polymer in biomedical applications. Prog Polym Sci. 2011; 36: 981-1014.
8. Lee KY, Mooney DJ. Alginate: properties and biomedical applications. Prog Polym Sci. 2012; 37: 106-126.
9. Hoffman AS. Hydrogels for biomedical applications. Adv Drug Deliv Rev. 2012; 64: 18-23.
10. Danhier F, Ansorena E, Silva JM, Coco R, Le Breton A, Préat PLGA-based nanoparticles: an overview of biomedical applications. J Control Release. 2012; 161: 505-522.
11. Makadia HK, Siegel Poly lactic-co-glycolic acid (PLGA) as biodegradable controlled drug delivery carrier. Polymers (Basel). 2011; 3: 1377-1397.
12. Hoare TR, Kohane DS. Hydrogels in drug delivery: progress and challenges. Polymer. 2008; 49: 1993-2007.
13. Volk T, Jahde E, Fortmeyer HP, Glüsenkamp KH, Rajewsky MF. pH in human tumour xenografts: effect of intravenous administration of glucose. Br J Cancer. 1993; 68: 492-500.
14. Carvalho C, Santos RX, Cardoso S, Correia S, Oliveira PJ, Santos MS, et al. Doxorubicin: the good, the bad and the ugly effect. Curr Med Chem. 2009; 16: 3267-3285.
15. Swain SM, Whaley FS, Ewer MS. Congestive heart failure in patients treated with doxorubicin: a retrospective analysis of three trials. Cancer. 2003; 97: 2869-2879.
16. Vermonden T, Censi R, Hennink WE. Hydrogels for protein delivery. Chem Rev. 2012; 112: 2853-2888.
17. Comsa S, Cimpean AM, Raica M. The story of MCF-7 breast cancer cell line: 40 years of experience in research. Anticancer Res. 2015; 35: 3147-3154.
18. Dash S, Murthy PN, Nath L, Chowdhury P. Kinetic modeling on drug release from controlled drug delivery systems. Acta Pol Pharm. 2010; 67: 217-223.
19. Yu LX, Kopcha M. The role of the pharmaceutical sciences in the FDA's quality by design program. Pharm Res. 2017; 34: 2688-2696.
20. Ventola CL. Progress in nanomedicine: approved and investigational nanodrugs. P T. 2017; 42: 742-755.
21. Fessi H, Puisieux F, Devissaguet JP, Ammoury N, Benita S. Nanocapsule formation by interfacial polymer deposition following solvent displacement. Int J Pharm. 1989; 55: R1-4.
22. Maeda H, Wu J, Sawa T, Matsumura Y, Hori Tumor vascular permeability and the EPR effect in macromolecular therapeutics: a review. J Control Release. 2000; 65: 271-284.
23. Dong Y, Feng SS. Methoxy poly (ethylene glycol)-poly(lactide) (MPEG-PLA) nanoparticles for controlled delivery of anticancer drugs. Biomaterials. 2004; 25: 2843-2849.
24. Ganji F, Vasheghani-Farahani S, Vasheghani-Farahani E. Theoretical description of hydrogel swelling: a review. Iran Polym J. 2010; 19: 375-398.
25. Kato Y, Ozawa S, Miyamoto C, Maehata Y, Suzuki A, Maeda T, et al. Acidic extracellular microenvironment and cancer. Cancer Cell Int. 2013; 13: 89.
26. Berger J, Reist M, Mayer JM, Felt O, Peppas NA, Gurny R. Structure and interactions in covalently and ionically crosslinked chitosan hydrogels for biomedical applications. Eur J Pharm Biopharm. 2004; 57: 19-34.
27. Higuchi Mechanism of sustained?action medication. Theoretical analysis of rate of release of solid drugs dispersed in solid matrices. J Pharm Sci. 1963; 52: 1145-1149.
28. Alexis F, Pridgen E, Molnar LK, Farokhzad OC. Factors affecting the clearance and biodistribution of polymeric nanoparticles. Mol Pharm. 2008; 5: 505-515.
29. Allen TM, Cullis Liposomal drug delivery systems: from concept to clinical applications. Adv Drug Deliv Rev. 2013; 65: 36-48.
30. Felt O, Buri P, Gurny R. Chitosan: a unique polysaccharide for drug delivery. Drug Dev Ind Pharm. 1998; 24: 979-993.