Synergistic effect of waste-derived β-tricalcium phosphate microbeads loaded in hydroxyapatite-keratin-polyvinyl alcohol composite matrix in drug release for osteosarcoma treatment

Background Sustained drug delivery system (DDS) for clinically relevant osteosarcoma medications is a promising strategy for treatment. β-tricalcium phosphate (β-TCP) microbeads loaded with doxorubicin hydrochloride (DOX) and cis-diamminedichloroplatin (CDDP) anticancer drugs in a matrix of hydroxyapatite-keratin-polyvinyl alcohol composite matrix scaffolds (HAp-K-PVA) was developed as promising DDS. HAp, β-TCP, and K utilized for the development of DDS were resourced from avian eggshells and human hairs, respectively, and duly characterized before application. Methods The β-TCP/alginate microbeads were fabricated using droplet extrusion and ionotropic gelation, and integrated into secondary drug carrier HAp-K-PVA composite matrix, via freeze gelation. The physicochemical and thermal characterization of developed microbeads and matrix scaffolds was performed. Results When DOX and CDDP were co-loaded in DDS, a synergistic impact was observed after 30 days of continuous release, in contrast to the immediate outburst as seen with individual DOX and CDDP releases. Besides, the drug release from the microbeads only, the release with the HAp-K-PVA composite matrix scaffolds was observed slower. The controlled release, antibacterial effectiveness against the test pathogens and cell viability with osteoblast-like osteosarcoma (UTOS) cells indicated the therapeutic potential for the treatment of osteosarcoma in situ. The cell viability was observed for 24 h, which showed nearly 90% after 24 h for HAp-K-PVA composite matrix scaffolds, decreased for all the scaffold groups after 72 h, indicating the enhancement due to combined synergistic effect of the co-loaded drugs. Conclusion This study established a promising foundation for novel and sustainable biomaterials for osteosarcoma treatment. Further advancement holds the potential to enhance patient clinical outcomes and foster advancements in the field of regenerative medicine.

of long bones, most often found in the proximal tibia, distal femur, and proximal humerus during the fast growth period of development.Among these, bone cancer is the 6th most frequent cancer occurring in children and adolescents, with a five-year survival rate mainly because of its tendency for local and distal recurrence [3].The annual incidence rate of OS globally is 2-4 million people aged 0-24 years (highest in Asia-Pacific region), 1.7 per million people aged 25-59 years (highest among Blacks), and 4.2 per million people > 60 years (highest among Whites), with males more frequently affected than females [4].Each year, 400 new instances of osteosarcoma are identified in the USA, whereas in Europe, the yearly incidence rate is between 10 and 26 per million [5].Further, despite of biomedical research and facilities, the survival rate has been below 70% for individuals with localized disease and below 20% for patients with metastases since decades [6].The study used a novel approach to address the increasing number of patients worldwide with the diseases mentioned in epidemiological data.
Osteosarcoma development is significantly influenced by the signaling elements of the bone microenvironment.BMP2 and TGFβ are circulating factors for promoting osteoblast development, osteosarcoma differentiation, and malignancy [7,8].The cellular components such as tumor-associated macrophages (TAMs) and environmental factors such as radiations and chemicals (radium, fluoride, chromium salts, beryllium oxide, asbestos etc.) interplay in the immune microenvironment for osteosarcoma development [9,10].
Osteoclasts that break the older or damaged osteocytes are regulated by RANK signaling mediated by RANKL protein expression.The dysregulation of RANK signaling due to released factors such as interleukins (IL) and TGFβ modulate RANKL expression on the osteoclast, reducing bone resorption, inhibiting osteoblast differentiation and osteocalcin expression, and promoting tumor progression [11,12].Osteosarcoma (OS) is driven by mutation in genes such as TP53 and RB1 tumor suppressor genes can induce osteosarcoma-like tumors in mesenchymal stem cells committed to the osteogenic lineage.Further, genes related to osteoblast development and the Wnt protein family play significant roles in osteosarcoma development [13].Epigenetic pathways regulate osteosarcoma tumor development via DNA methylation, histone modifications, and involvement of non-coding RNA molecules [14].miRNAs play a role in controlling the development and advancement of osteosarcoma by influencing cell proliferation, invasion, metastasis, and apoptosis [15].
Patients suffering from osteosarcoma have subsequent bone mass deterioration leading to a wide range of skeletal problems including bone pain, swelling, bone fractures, and hypercalcemia.The differentiation of mesenchymal cells, loss of tumor suppressor genes, activation of oncogenes, epigenetic changes, and production of cytokines are the primary causes of osteosarcoma standard treatments involving chemotherapy and irradiation; both can cause harm to the healthy tissues because of high-on-drug concentrations inside the body such as those produced by intravenous injection, causing systemic toxicity and necrosis of physiological organs, including jaw osteonecrosis [16].miRNAs are still being investigated through in vitro studies on osteosarcoma cells [15].The first line of treatment for malignant osteosarcoma is neoadjuvant chemotherapy using the MAP regimen (methotrexate, Dox, and CDDP), followed by surgical and radiotherapy.These strategies have limitations such as side effects or reoccurrences of disorder due to incomplete removal of OS tissues [17].Osteosarcoma cells often develop resistance to commonly used chemotherapy drugs, reducing the effectiveness of treatment and leading to relapse [18].DOX-mediated synergistic chemo-photodynamic therapy with C. vulgaris effectively halted tumor progression in an orthotopic osteosarcoma mouse model by promoting tumor cell apoptosis and inhibiting tumor proliferation and angiogenesis [19].Additionally, DOX-loaded octacalcium phosphate complexes with Fe 3+ and Cu 2+ ions facilitated DNA binding and ROS production, increasing cytotoxicity, though Cachannel blockers could sensitize cells to DOX and reduce its efficacy [20].
In this direction, a novel therapeutic approach that is both with least harmful side effects and the most effective against cancer is thus required for the treatment of osteosarcoma.Drug delivery carriers like hydrogels, micro-and nanoparticles, liposomes, biodegradable polymers, and calcium phosphates that deliver large doses of medicine with controlled release directly to the tumor site with better outreach, have the potential to reduce or eradicate local recurrence of bone cancer after marginal excision to comfort the patient.However, biodegradable polymer-based DDS alone may be harmful to the loaded pharmaceuticals or the surrounding tissues due to the disintegrated fragments, acidic byproducts, or the harsh solvents essential for their breakdown [21].A comparable study demonstrated that when hydroxyapatite (HAp) is combined with doxorubicin (DOX) within a polyvinyl alcohol (PVA) matrix (forming a DOX-HAp-PVA nanocomposite), it exhibited cytotoxic effects on osteosarcoma cells (MG 63).This combination not only enhanced the physicochemical properties, but also led to improved biological properties [22].Achieving optimal drug release kinetics is crucial, but burst release or inefficient release can lead to systemic toxicity or suboptimal concentration respectively, at tumor sites.However, controlled release of such pharmaceuticals without risking the quality of the delivered product and the damage to the tissues at the site of treatment, the ceramic-based DDS in a two-stage delivery seems promising.This motivated to develop calcium phosphate (CaP)-based microbeads loaded in HAp-K-PVA composite DDS for osteosarcoma treatment.However, they also present limitations such as potential toxicity, complex manufacturing processes, and challenges in achieving controlled drug release.
CaP, most effectively administrated intravenously, follows a uniform drug release and may be used to treat osteosarcoma with better cytostatic regulation.In 2023, Aaddouz et al. found that pharmaceuticals, including anticancer drugs, are delivered due to apatite's strong surface contact capabilities and their ability to bind neutral, positively, and negatively charged molecules [23].Further, the CaP-based local DDS may reduce the frequency of injections, improve patient's quality of life; and halt the progression of osteosarcoma by restricting high systemic drug concentrations [24].Because of these, CaP ceramic as a major filler for bone abnormalities and as a means of delivering medications is promising.Its high surface area to volume ratio greatly impacts cell-biomaterial interaction as its ultra-thin structure is remarkably similar to the microstructure of biological apatite [25].Since the sintered HAp is a bioceramic and is mechanically not strong enough to be employed as a biomaterial, combining with natural or synthetic polymers such as K and PVA may impart structure stability while improving processability and biocompatibility of matrix [26].K, a potential biomaterial is an endogenous protein exhibiting intrinsic biological activity, excellent degradability, and biocompatibility and has acquired a great deal of attraction as a regenerative medicine, biomimetic scaffolds, and bone tissue engineering.It has been demonstrated that K porous scaffolds facilitate pre-osteoblast cell adhesion, proliferation as well as development due to the presence of cell-binding motif sequences like RGD, LDS, and EDS [27].Despite its potential as a biomedical material, its poor hardness and stress-induced malleability make it difficult to achieve the desired mechanical properties [28].
Patients with osteosarcoma often undergo a dual-phase treatment involving DOX and CDDP-based chemotherapy, recognized as a gold standard regimen [29].DOX, a potent anti-neoplastic agent, hampers nucleic acid synthesis and disrupts topoisomerase II function, demonstrating significant efficacy against osteosarcoma.Cisplatin, by interacting with the cellular DNA-N7 subunit, induces apoptosis in osteosarcoma cells, making it a crucial component in the therapeutic strategy [30][31][32].The combined use of these medications has demonstrated clinical efficacy, exhibiting increased cytotoxicity at lower doses and addressing drug resistance [33][34][35][36].The study focused on examining the possible use of these scaffolds as depots for osteosarcoma cytostatic agents.

Materials
All the reagents and chemicals were purchased analytical grade from Sigma-Aldrich.Sodium phosphate monobasic monohydrate, sodium phosphate dibasic (0.1 M), cisdiamine platinum (II) dichloride (cisplatin, CDDP, 99.9%, trace metals basis, lot), doxorubicin hydrochloride CRS (DOX) (code D2975000, EPRS, France), sodium alginate, and calcium chloride dihydrate LR.Sodium dodecyl sulfate, PVA (Mol wt.145,000 g/mol, 98% hydrolyzed, Sigma-Aldrich), was bought from the German company Merck.These chemicals and reagents used in the study were not purified before usage.For the cell culture studies, the laboratory chemicals of clinical grade were used.

Synthesis of β-TCP and hydroxyapatite from eggshells
Chicken eggshells collected from the institute canteen, washed for 30 min at 100 °C in distilled water were dried for 30 min at 80 °C [37].Following drying, the cleaned eggshells were subjected to a 4 h calcination process at 1100 °C to convert inherent carbonated calcium (CaCO 3 ) to calcium oxide (CaO).After being calcined, the eggshells were finely ground using a mortar-pestle and sieved for sorting fine powder.The powder weighed 11 g followed by mixed in 50 mL of distilled water to convert into calcium hydroxide (Ca(OH) 2 ).Further, it was dissolved by adding 7.15 ml of 1 M phosphoric acid and agitating the mixture for 1 h.The pH was adjusted to 10 by adding 30% NH 4 (OH) 2 , and the mixture was agitated constantly for 5 h to achieve a thick suspension.Furthermore, this suspension was dried in an oven for 24 h at 60 °C.β-TCP and HAp were effectively synthesized and characterized by X-ray diffraction (XRD) from the waste eggshells and stored in a desiccator for further study (Fig. 1A).

Extraction of K from waste-derived human hair
K was extracted from human hair using the Shindai method [38].Briefly, the hair samples were thoroughly and washed using a 1% w/v detergent solution.After that, the sample was immersed in 60% v/v ethanol for 5 min for sterilization followed by rewashing with distilled water and dried overnight.It was then subjected to delipidization for 24 h in methanol and chloroform (2:1 v/v) at their azeotropic temperatures.After 24 h, the fat globules were drained off using a sieve and rinsed thoroughly with distilled water.The samples were left out to dry overnight and a lysis solution was prepared using 6 g of sodium sulfite, 1 g of SDS, 200 mg of urea in 100 ml of distilled water, and 10 g of hair samples were incubated in this solution for 16 h at 60 °C followed by vacuum filtration.The filtrate was then centrifuged at 10,000 rpm (Eppendorf 5810R) for 30 min and dialyzed supernatant using a 12-14 kDa snakeskin membrane (Sigma-Aldrich), against a 0.05 M PBS solution at pH 7.2 for 48 h using an automated dialysis system developed by our group (buffer changed automatically achieving threshold TDS value) [39] (Fig. 1B).

Preparation of β-TCP and alginate microbeads
β-TCP beads were prepared using liquid ejection and ionotropic gelation where the water-based solution containing 0.7 wt% of sodium alginate, 30 wt% of silica sol, and 0.2 wt% sodium citrate were constantly mixed at 1000 rpm, while β-TCP was gradually added.The suspension was homogenized for 15 min with a Branson ultra-sonicator (Digital Sonifiei 450) (150 W power) to disintegrate any potential agglomerates.The suspension was then injected using a syringe (3 cc; 0.45 mm needle diameter) into a cross-linking solution containing CaCl 2 (0.1 mol/L) in ddH 2 O/ethanol (80/20 v/v) solvent solution was kept with the microbeads for a period of 18 h at RT and were further rinsed with ddH 2 O thrice to eliminate excess calcium ions that might have accumulated on beads surface.The beads were decanted and then dried in a desiccator for the next 3 days.The stored microbeads were used for further study (Fig. 1C).

Cytostatic drugs loading into microbeads
β-TCP beads were incubated in concentrated solutions of cytostatic drugs containing 60 µg/mL CDDP and 60 µg/ mL per DOX in ddH 2 O.To incorporate DOX and CDDP in 0.5 mL solution, microbeads weighing approx.0.055 g of microbeads and 4 g of microbeads-loaded matrix of each group were incubated for 48 h at a temperature of 37 ºC with a constant stirring of 1000 rpm and neutral pH.A cytostatic stock solution was added to 0.055 g of beads to achieve uniform drug loading.The supernatants of DOX and CDDP were collected at the incubator and centrifuged at 15,000 rpm for 15 min to eliminate decontamination, respectively.The spectroscopic concentration of loaded DOX was determined over different time intervals of 5 min exponentially to 48 h.

Preparation of hydroxyapatite-K-PVA (HAp-K-PVA) composite scaffolds loaded with drug-loaded microbeads
Porous HAp-K-PVA composite scaffolds were fabricated using freeze gelation by incorporating DOX and CDDPloaded microbeads into the matrix and by adding these cytostatic drugs directly to the scaffold as control.To prepare the composite scaffold, a homogeneous water-based solution was prepared using HAp (1 wt%), PVA (12 wt%), silica solution (1.98 wt%), and citric acid (1.93 wt%) with a final pH adjusted to 8 by ammonia.The beads and matrix suspension were uniformly dispersed into cylindershaped polyvinyl chloride (PVC) molds.No crosslinker was added while incorporating microbeads in the cytostatic solutions.The different possible combinations with the selected materials are proceeded for the study of drug release.Thus, the developed different sample groups (Table 1), DOX Matrix (DOX loaded in composite matrix); CDDP Matrix (CDDP loaded in composite matrix); Simple Matrix + DOX Beads (DOX-loaded microbeads in composite matrix); Simple Matrix + CDDP Beads (CDDP-loaded microbeads in composite matrix); DOX Matrix + DOX Beads (DOX-loaded microbeads in DOX-loaded composite matrix); CDDP Matrix + CDDP Beads (CDDP-loaded microbeads in CDDP-loaded composite matrix); Simple Matrix + (DOX + CDDP) Beads (DOX and CDDP co-loaded microbeads in composite matrix); DOX Matrix + (DOX + CDDP) Beads (DOX and CDDP co-loaded microbeads in DOX-loaded composite matrix); and CDDP Matrix + (DOX + CDDP) Beads (DOX and CDDP co-loaded microbeads in CDDPloaded composite matrix), were studied for release kinetics, and cytotoxicity with an unloaded composite matrix as a reference.The different sample groups were then uniformly placed in 10-mm-diameter by 5-mm-high polyvinyl chloride (PVC) molds.After freezing for 20 min at -150 °C, the molds went through seven cycles of freezing and thawing cycles.The molded samples were dried and stored in a desiccator at RT, for further characterization (Fig. 1D).

Physiochemical characterization
The functional groups of the chemical on the sample are acquired using the Fourier-transform infrared spectroscopy (FTIR) spectroscopy (Buker Alpha II spectrometer, Germany), in the transmittance range 400-6000 cm −1 with a resolution of 4 cm −1 and 6 scans.XRD was done using PANalytical B.V. Lelyweg 1 7602 EA Almelo, Netherlands.The diffraction data were acquired at a 2Ө angular range of 10°-90° with CuK radiation (λ = 0.154 nm).The XRD machine was operated at an accelerating voltage of 35 kilovolts (kV) and a current of 20 milliamperes (mA), with a scanning rate of 3 degrees per minute (°/ min) at a 2θ angle.
Field-emitting scanning electron microscope (FESEM) images were obtained using Sigma 500 VP field-emitting scanning electron microscope (ZEISS Gemini 1), at 700X magnifications and an accelerating voltage of 3.0 kV and 5 mm working distance.EDAX APEXTM EDS software was used to carry out energy-dispersive X-ray spectroscopy (EDS) to qualitatively investigate the elemental compositions of the samples.The thermal behavior of developed samples was studied with thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC).Thermal profiling of the synthesized sample was conducted using a thermogravimetric instrument (Perki-nElmer TGA 4000) and DSC (PerkinElmer DSC 6000).The measurements were carried out under N2 (20 mL/ min) conditions with a heating rate of 10° C/min, ranging from 28 to 600 °C.Particle size and zeta potential analysis was performed by Zetasizer Nano ZS90 (Malvern).Surface contact angle measurement with a drop shape analyzer (DSA100, KRU SS) was used to gauge the surface wettability of the HAp-K-PVA composite matrix, a sessile drop of DI water was placed, and the contact angle was captured and calculated.

Antimicrobial activity test
An antimicrobial test was conducted to assess the antibacterial efficacy of HAp-K against Staphylococcus aureus (S. aureus) and Pseudomonas sp.Bacterial strains of S. aureus and Pseudomonas sp. were inoculated into 10-ml luria broth (LB) media from the stock bacterial culture and grown overnight.The culture was maintained at 37 °C in a BOD incubator for bacterial growth.This culture served as the basis for antimicrobial testing using the disk and well diffusion method.The OD of overnight S. aureus and Pseudomonas sp.culture was adjusted to 0.5 McFarland Scale.The sterile agar surface of a Luria agar plate was evenly inoculated with cultures of S. aureus and Pseudomonas sp.(forming a bacterial lawn).Impregnated disks with various concentrations of the composite solution were placed in the center of respective plates (well diffusion method), and the matrix composite was positioned in the plate (disk diffusion method).After incubating for 24 h at 37 °C, the plates were examined for bacterial growth.The antibacterial activity was indicated by a confluent lawn of growth with consistently round zones of inhibition.The diameter of the disk zones, representing total inhibition zones, was measured to the nearest full mm.

Drug release kinetics
In the drug release study, a predetermined quantity of drug-loaded microbeads was introduced in 1 ml Tris-HCl buffer (0.1 mol/L) under continuous stirring with pH 7.4 at 37 °C.Samples of the released media were collected at different time intervals while ensuring little disturbance to the microbeads.It was assumed that drug release followed a consistent pace throughout time.The collected medium was then replaced with a fresh quantity of buffer in the beaker.The obtained samples were evaluated by measuring the OD at λ = 390 nm for CDDP and 480 nm for DOX (UV Spectrophotometer) to measure the amount of DOX and CDDP released.The supernatants of DOX and CDDP were collected at the incubator and centrifuged at 15,000 rpm for 15 min to eliminate decontamination, respectively.The spectroscopic concentration of loaded DOX was determined over 90 min in a semi-micro-cuvette where 1 mL of DOX and CDDP solution was mixed with 0.055 g of β-TCP beads in an interval of every 5 min.The release of DOX and CDDP from the beads/matrix scaffold composites was observed for 30 days in a 1 ml Tris-HCl buffer (0.1 mol/l, initial pH of 7.4) at 37 °C with constant shaking.As a control, unloaded microbeads/matrix composites were taken, and all samples were measured in triplicates (n = 3).

In vitro analysis and cytotoxicity
The biocompatibility of HAp-K-PVA composite scaffolds cultured with U2OS cell line having characteristics like human osteoblast was assessed by the MTT assay at Institute of Nanoscience and Technology, Mohali, India, with approval of Institutional Ethics Committee (IEC).
The cytotoxicity of the samples was evaluated using the MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) test on U2OS cells.In a 96-well plate, the cells were seeded at a density of 5X10 4 cells/well with 100 µL of Minimum Essential Medium Eagle (MEM) containing composite scaffold and microbeads samples (0.055 g), 10% fetal bovine serum, and 1% penicillin-streptomycin, and then incubated at 37 °C and 5% CO2.The growth media was replaced after 24 h with samples.After the 24 h of incubation period, 50 µL of MTT solution (1 mg/ ml) was added to each well and incubated for another 2 h.After removing the MTT solution, the formazan produced by the metabolic breakdown of MTT by living cells was dissolved by adding isopropanol.An automated microplate reader (BioTek Cytation 5) was used for measuring formazan absorbance values at 570 nm, for process quantification.It was repeated on day 3 for assessing cell proliferation.The MTT experiment was conducted in triplicates for each sample (n = 3).

Characterization of eggshell derived β-TCP and HAp
XRD analysis at 1100 °C during the calcination process of raw eggshell reveals the formation of biphasic CaO and Ca(OH) 2 , resulting in the production of CaCO3 as shown in Fig. 2A.In the crucial calcination phase, the carbon component in CaCO 3 decomposes to emit carbon dioxide (CO 2 ) leaving behind calcium and oxygen molecules (CaO) for utilization in HAp production.Once the CaO absorbed moisture from its surroundings, it leads to the formation of Ca(OH) 2 in the sample as confirmed with XRD peak at 34.1°.The intensity of peaks for both hydroxyapatite (HAp) and β-tricalcium phosphate (β-TCP) is significantly higher up to 1100 °C, indicating the efficient formation of finely ground HAp crystals with β-TCP as a secondary phase.The resulting HAp product is particularly well suited for bone replacement due to its osteoconductive and bioresorbable properties attributed to HAp and β-TCP, respectively [40].Several phase transformations occur during HAp calcination at 1100 °C due to the high temperature.The first significant change during the calcination process is the removal of water molecules from the hydroxyapatite structure.
Water molecules bound within the hydroxyapatite lattice are driven off, leading to the dehydration of the material.Hydroxyapatite begins to decompose into several phases.The primary decomposition products include tricalcium phosphate (Ca 3 (PO 4 ) 2 ), tricalcium phosphate (Ca 4 P 2 O 9 ), and CaO.These phases are formed through the breakdown of the calcium phosphate backbone of hydroxyapatite, and it undergoes further transformation, and β-TCP begins to form.The crystallinity of the resulting phases increases at 1100 °C, which promotes the rearrangement of atoms and the growth of crystal domains, leading to the development of well-defined crystalline structures in the final products.
The FTIR study relied on absorption bands associated with vibrations of functional groups within the molecules.The FTIR spectra, presented in Fig. 2B, illustrate the analysis results of the synthesized sample that underwent calcination at 1100 °C.After the calcination of raw eggshells, the peak appeared at 1455., which were generated at frequencies ranging from around 870 to 962 cm −1 .The low quantity of CO 3 −2 impurities in those samples was inferred from the observation of faint peaks in their spectra.The CO 3 −2 v3 vibration band, which was produced by asymmetric stretching, peaks at 1450 cm −1 .The FTIR spectra showed that the presence of PO 4 3− represented a structural link between HAp molecules.The FTIR spectra of biphasic HAp and TCP were devoid of the OH absorption band, which differentiates them from the HAp phases at temperature 1100 °C.The carboxylic acid peak, arising from the stretching vibrations of the C=O bond and the bending vibrations of the O-H bond, typically appears in the range of 1700 cm −1 to 1300 cm −1 in the FTIR spectrum.The exact position of this peak can be influenced by the molecular environment of the carboxylic acid group, intermolecular interactions, and chemical interactions with other functional groups.The thorough spectroscopic investigations, encompassing FTIR, yield a precise comprehension of the produced material's phase composition and intermolecular interactions.Shifting in the carboxylic acid peak might be attributed to the changes in the chemical environment or interactions, such as hydrogen bonding, and may also result from alterations in the protonation state of the carboxylic acid group.
The size of the particles in the calcined raw eggshells is more consistent between 150 to 300 nm by FESEM analysis.It was observed that the agglomeration of particles took place after the chemical precipitation and second calcination processes, which in turn caused the creation of bigger particles in a range of sizes.The particle agglomeration in the composite matrix reduced overall homogeneity and enlarges particle size and dimension distribution.This caused porosity, surface area, and mechanical strength variations, potentially creating void spaces that could affect permeability, surface area, and drug loading capacity, critical for drug delivery.The mechanical properties of the K-PVA composite matrix also influenced by particle agglomeration.While smaller, dispersed particles enhance mechanical strength the larger agglomerates, like those in biphasic HAp and TCP, can act as stress concentrators, potentially resulting in reduced mechanical performance.Similar irregular particle structures were discovered in previous HAp-derived from eggshell investigations as well [41].According to Goloshchapov et al., rising temperatures cause particles to aggregate and enlarge the particle size (Fig. 2D).High-resolution transmission electron microscopy (HR-TEM) imaging of HAp extracted from eggshells calcined at 1100 °C reveals significant crystalline growth and the transition to the β-TCP phase by altering the crystal structure.These changes impact the mechanical properties, interface structure, bonding, and bioactivity of HAp, thereby affecting its interactions with biological systems.Modifications in drug release kinetics, crystal structure, and surface chemistry influence tissue responses.The atomic arrangement in the HAp crystal lattice may be seen as fringes in HR-TEM pictures indicating a few layers and sharp edges [42].The crystal structure of HAp is hexagonal.HAp grain size, shape, and the identification of nanoscale features like nanoparticles or nano-sized holes can be determined through HR-TEM analysis.Calcination at high temperatures may have caused grain development, producing bigger crystalline domains in HAp, which can affect its mechanical properties, bioactivity, surface area, and interactions with cells or other biomaterials (Fig. 2C).
The EDS analysis of the raw eggshell after calcination (Fig. 2E) revealed Ca and O as the primary components.This elemental composition aligned with the CaO and Ca(OH) 2 compounds identified through XRD analysis.
The introduction of H 3 PO 4 during the chemical precipitation process, along with the addition of phosphate ions, led to the incorporation of phosphorus elements in the samples.The zeta potential of nanoscale HAp is the electromotive force (EMF) between the molecule's surfaces and the external fluid.Several variables, including the solution's pH, the particle's surface charges, and the presence of other ions or molecules, might influence nano HAp zeta potential.The zeta potential value of HAp sample powders is shown in (Fig. 2G).The FESEM data indicates a tendency for nanoparticle agglomeration and cluster formation, as evidenced by the larger hydrodynamic particle size values (Fig. 2F).The maximum negative zeta potential (− 19.7 ± 10.1 mV) which is in the threshold level with a polydispersity index of 0.183.Managing particle aggregation is crucial for maximizing drug release rate and therapeutic effectiveness of DDS.An early burst out release from smaller particles followed by a protracted lag phase results in the agglomeration faster than larger particles.The requisite of efficient drug delivery is a maximum surface area that enhances interactions with cells and biomaterials.This is essential for achieving prolonged and targeted release of chemotherapeutic drugs at the tumor location.The UV spectrophotometric analysis.

Characterization of human-derived K
The FTIR analysis revealed the chemical composition of the purified K showing typical characteristic peaks located at 1539 cm −1 , which is consistent with the bending distortion of the C-NH bond.The amine group N-H stretching vibrations are 3415 cm −1 , while the amine group O-H flexing liberation peak at 3523 cm −1 [43].Amide I is 1650 cm −1 and is associated with the stretching vibration of C=O bonds; Amide III is 1232 cm −1 and is formed from C-N stretching and N-H bending; and the transmission bands of peptide bonds (-CONH) are linked to the stretching vibration of N-H and O-H.FTIR spectroscopy demonstrates that their closeness to hydrophilic and hydrophobic molecules makes them soluble in both aqueous and organic fluids (Fig. 3A).The FTIR analysis results are consistent with a comparative study done by Agarwal et al., where they obtained the characteristic medium absorption peak for Amide A vibrations observed at 3276 cm −1 [44].This peak originated from primarily the N-H stretching vibrations but also is contributed by the Fermi resonance from the first overtone amide II.The strong amide I band was observed at 1640 cm −1 , associated with the C-O stretching vibrations; a strong peak at 1517 cm −1 (amide II) has been assigned to N-H bending and C-N stretching vibrations.A weak band at 1234 cm −1 was also observed and assigned to amide III with contributions from C-N and C=O stretching and N-H bending vibrations [44].Overall, the strong amide band from sulfides of keratin molecules was observed.The existing functional groups, along with amide bands, could connect with other compositions of DDS to form a continuous matrix.The details about the crystallographic phases of K were analyzed by XRD.The lyophilized extract produces an XRD pattern with a broad peak at 9.72°, indicating the presence of both α-helix and β-sheet structures.Additionally, a more prominent peak at around 21.8° (2θ) was identified, which was attributed to the β-sheet structure.The extract K is amorphous and does not crystallize under normal circumstances.The reading that was acquired agrees with those that were given in earlier K studies [45] (Fig. 3B).
The UV spectrophotometric analysis of the K extract revealed an absorbance maximum of 270 nm, consistent with previous research on protein absorbance maxima and K extraction.The deviation in UV absorbance spectrum of keratin standard samples could indicate alterations in its protein secondary structure, such as α-helix, β-sheet, or random coil conformations.Protein function, including drug interaction, can be affected by structural changes [46].Due to their rigidity, α-helices may impede drug release by requiring a longer time for drug molecules to dissolve.β-sheets' stability may balance drug release, binding strength, and accessibility.Drug release may be faster in the less stable random coil shape because loosely bound medicines split more easily [47].With K constituting 95% of human hair and lipids comprising the remaining 5%, it is reasonable to infer that the extracted protein is indeed K. To confirm the results, nevertheless, another study was conducted.Following the confirmation of the presence of K in the hair extract, the Lowry assay was conducted to determine the concentration of K in the extract.Results from the Lowry assay, corroborated with the results of dry weight analysis after lyophilization, reveal an approximate concentration of 39 mg/mL of K in the extract (Fig. 3C, D).

Characterization of drug-loaded β-TCP microbeads
The synthesis of biphasic CaO and Ca(OH) 2 is revealed by the XRD pattern (Fig. 4A) of fresh eggshell that has been calcined at 1100 °C.Upon analysis, the synthesized composite was identified to contain HAp in conjunction with β-tricalcium phosphate (β-TCP).Hap shows 28°, 32.34°, 39° (2θ) and β-TCP shows 34.41° (2θ).Peaks for both HA and β-TCP were more intense up to 1100 °C (Fig. 4A), demonstrating the efficiency of this temperature in producing very crystallized HA powder with β-TCP as a secondary phase.The peak at around 10°-15° corresponds to CDDP, the peak in the microsphere coloaded with DOX, and CDDP was lesser intensified due to the bonding between the drugs.Further inferred that the interfacial bonding caused by the contracting polymeric scaffold composite matrix may have caused compression.A peak of HAp that emerged at 56.76° co-loaded with cytostatic for HAp-K-PVA XRD plot and nearly at 55.80° [22].XRD peak shifts in TCP microbeads can signify phase alterations, encompassing α-TCP, β-TCP, and amorphous phases contingent upon synthesis and processing parameters.These shifts, indicative of changes in phase proportion, are influenced by factors such as processing techniques, temperature, and mechanical stresses, which can induce lattice distortions or phase transitions.Furthermore, the interaction of organic (doxorubicin) and inorganic (cisplatin) compounds with hydroxyapatite crystal structure in microbeads can disrupt lattices or modify crystallite size, resulting in XRD peak shifts.The binding of these anticancer medications with hydroxyapatite can induce alterations in its crystal structure, manifesting as new peaks in the composite material, reflecting changes in atom composition or rearrangement.The co-loading of doxorubicin and cisplatin in HAp microbeads may prompt phase transformations or modifications in crystalline phases, potentially leading to peaks corresponding to both medications, albeit with varying locations depending on their polymorphic forms.The study conducted by Martin et al. indicates that changes in crystallinity and molecular interactions, as evidenced by XRD peak shifts, can significantly impact the release rates of drugs from microbeads.It has been observed that amorphous drugs in polymer matrices exhibit faster release rates than their crystalline counterparts [48].
The functional groups of the microbeads were analyzed using FTIR spectra, which are relevant with HAp and K as major constituents (Fig. 4B).The bands at 3570 and 632 cm −1 correspond to the stretching and bending vibrations of the hydroxyl group, respectively, and the absorption bands at 1092, 1035-1031, 961, 603, 565, and 470 cm −1 correspond to distinct vibration modes of the phosphate group (PO 4 3− ) in HAp.From the FTIR spectra of drug-loaded composite scaffolds HAp-K-PVA, unique peaks were discovered at wave numbers 669.53  ) distinctive absorption bands were found at 1482, 1452-1460, 1421-1421, and 874-884 cm −1 because of the absorption of CO 2 .The spectra also revealed evidence of interactions between molecules between microbeads and medicines in the form of a shift in the peak of carboxylic acid from 1641 to 1622 cm −1 .The HAp-K-PVA FTIR spectra revealed a band at 2043.54 cm −1 , which might be the result of an overlap between 2077.63 cm −1 of HAp and 2085.67 cm −1 of DOX.The peak was observed as the single bond CH 2 single bond asymmetric stretching band of PVA at 2947.23 and 831.31 cm −1 .The peak corresponds to the CH 2 single bond group of the PVA molecule's asymmetric bending vibration at 1384 cm −1 and structural OH at 1618 cm −1 which corresponds to 1381.03 and 1613.68 cm −1 .Additionally, the spectra revealed the usual CDDP peaks at 1600-1500 cm −1 (asymmetric amine bending) and 1300-1200 cm −1 (symmetric amine bending).According to Chen et al., peaks at 1285 cm −1 (C-O-C stretching) and 1407 cm −1 (C=C stretching), 1442 cm −1 (coupling of C N and N H) and 996 cm −1 (CO stretching) are the result of CDDP and DOX conjugation to scaffolds.The FTIR analysis revealed no significant peak shifts, indicating that doxorubicin and cisplatin effectively bond with the K-PVA composite matrix for co-delivery.This interaction between the anticancer drugs and the matrix enhances the stability of the drug-loaded system, thereby preventing premature release during storage or transportation.Solid hydrogen and ionic bonds enhance the encapsulation efficacy and reduce drug leaching.These interactions are essential for the maintenance of therapeutic levels over protracted periods, as they facilitate the establishment of a controlled and sustained drug release profile.
The amount of weight loss and heat stability of drugloaded microbeads were evaluated by TGA (Fig. 4C).The heating rate of 10˚C/min was employed up to 1400 °C temperature in the air atmosphere.The microbead was heated from 29 to 575 °C, and a weight reduction occurred from 99.8 to 85.2%.It was observed that there was a gradual weight loss for reference microbead sample showing from 99.816 at 29.5 °C which is due to the evaporation of absorbed water, then as the temperature increased to 424.62 °C, weight loss was 88.252% and eventually there was slow and gradual weight loss till 85.843 at 575.27 °C.It was observed that the microbead sample was found to be thermally stable, as shown by the steady curve seen across a wide temperature range.For CDDP-loaded microbeads, the temperature ranges from 29.54 to 575.46 °C corresponding to initial exponential weight loss followed by a gradual weight loss from 84.72 to 36.07%.The remaining inorganic mass may be attributed to the coupled groups produced between the microbead and CDDP.For DOX-loaded microbeads, the temperature ranges from 29.53 till 575.48 °C corresponding to initial exponential weight loss followed by a gradual weight loss from 99.691 to 69.046%.Almost following a similar weight loss, for the co-loaded microbeads, the temperature ranges from 29.62 till 575.33 °C corresponding to initial exponential weight loss followed by a gradual weight loss from 896.86 to 76.325%.The TGA analysis revealed that the microbeads containing the anticancerous drugs showed constant thermal stability over a broad range of temperatures, with a steady decrease in weight.The weight reduction patterns exhibited small variations across each drug-loaded sample, displaying distinct thermal characteristics.
DSC was utilized to analyze the temperature behavior of microbeads that were filled with anticancer cytostatic.The DSC thermogram (Fig. 4D) showed a steady rate of 10 °C/min of heat flow from 30 to 440 °C.The DSC plot was used to evaluate information regarding thermal events or transitions within the sample as a function of temperature in the context of microbeads co-loaded with DOX and CDDP.Microbead components, such as β-TCP and alginate, may alter the thermal behavior of HAp via interactions with the substance.The phase transition from crystalline to amorphous occurred in HAp at certain temperatures and the loaded drugs resulting in altered peak shapes might influence these variations in transitions.The endotherm, with a high of 105 °C on the DSC curve, shows that water was removed from the sample.During HAP crystallization, chemically bound water was released, accounting for the large peak of about 177 °C.The DSC analysis revealed the interactions between components, including β-TCP, alginate and HAp in drug-loaded microbeads and its phase transitions which represents water loss occurred at 105 °C and chemically bonded water release during HAp crystallization at around 177 °C were endothermic peaks on the DSC thermogram.Its shows that drug loading alters microbead thermal behavior.The observed changes in melting temperatures and crystallinity indicate significant interactions between the polymer and drug with the existence of drug amorphous forms.By comprehending these thermal features, the stability and effectiveness of drug-loaded microbead systems in drug delivery is assured.
Non-sintered β-TCP beads were approximately spherical in scanning electron micrographs, but their surface was partly structured, wrinkled, and rough as seen in (Fig. 4E).Cross sections of the beads revealed a very porous interior encased in a relatively thin but more compact outside shell which is favorable as it allows tissue development on bone implants within the body [50].
The densified regions are artifacts caused by sectioning while characterization.The pore size of the non-sintered microbeads was analyzed using ImageJ Software, and the average range of different microbeads comes in the range of 0.02-2043 µm.The size of the pores significantly influences both the efficacy of drug delivery and the drug release rate [51].Microbeads with very small pore sizes exhibited limited drug loading capacity due to restricted pore space, which impedes efficient drug encapsulation.Conversely, medium-sized pores offer an optimal balance between surface area and drug loading capacity, facilitating more controlled and sustained drug release.This leads to more predictable and consistent release profiles.The varying pore diameters affect drug release: smaller pores hinder encapsulation, medium pores balance space and surface area, and larger pores enable more drug but faster diffusion.
According to the EDS analysis, the primary constituents are Ca, O, and C, with Si and P coming in second and third place (Fig. 4F).These findings suggest that the first group of components may be related to the major component of the eggshell (CaCO 3 ), while the second group of elements may be related to the organic material present in the egg's structure.The cytostatic microbeads had an elemental composition that was comparable to that of the reference microbeads, at least in terms of the predominant components.While microbeads loaded with DOX had a weight proportion of Cl and microbeads treated with CDDP included a weight percentage of Pt, microbeads loaded with both medicines had both Cl and Pt in their elemental makeup.The EDX test reveals a Ca/P ratio of roughly 1.42 to 1.51, which is less than 2 and acceptable.The optimum Ca/P ratio for HAp is 1.67 [52] (Table 2).The β-TCP microbeads exhibited a calcium-to-phosphorus (Ca/P) ratio lower than the stoichiometric ratio of hydroxyapatite (HAp), which is 1.67.This suggests a calcium deficiency in relation to phosphorus.Given a Ca/P ratio of 1.45, the β-TCP microbeads may change their mechanical characteristics [53].This is because calcium is essential for maintaining hydroxyapatite's structural integrity and stability, and it can also influence the biomineralization process.The control over the temperature, pH, drop acceleration while adding phosphate solution and applying other treatment conditions such as ultrasonication or surfactant greatly affects the reproducibility of β-TCP and HAp [54].

Characterization of HAp-K-PVA composite matrix
The functional groups were analyzed by FTIR spectra (Fig. 5A).The typical PVA absorption bands were visible at 2940 cm −1 (CH 2 asymmetric stretching), 1408 cm −1 (CH 2 scissoring deformation), 1740 cm −1 (C=O from non-hydrolyzed ester groups and C-O stretching, respectively), 1634 cm −1 (adsorbed water), 1268 cm −1 (H-C-H bending in CH 3 ) and 845 cm −1 (C-C stretching).The transmission bands of the peptide bonds (-CONH) due to the presence of K connected to the stretching vibration of N-H and O-H, Amide I as 1650 cm −1 related to the C=O stretching bonds, and Amide III as 1232 cm −1 derived from C-N stretching and N-H bending, these bonds help optimize the scaffold preparation.Consequently, the absorption band at 1740 cm −1 merged into neighboring frequency ranges.The absorption bands at 1092, 1035, 961, 603, 565, and 470 cm −1 correspond to various vibration modes of the phosphate group (PO 4 3− ) in HAp, whereas the bands at 3,570 and 632 cm −1 correspond to the stretching and bending vibrations of the hydroxyl group, respectively.Due to the absorption of CO 2 , carbonate group (CO 3 2− ) characteristic absorption bands were observed at 1482, 1452-1460, 1421-1421, and 874-884 cm −1 [55].Additionally, the spectra revealed a peak shift of carboxylic stretch (CO) from 1641 to 1622 cm −1 , which indicated intermolecular interactions between scaffolds and drugs.The spectra also showed the typical CDDP peaks at 1600-1500 cm −1 (asymmetric amine bending) and 1300-1200 cm −1 (symmetric amine bending).CDDP and DOX conjugation to scaffolds is responsible for the appearance of peaks at 1285 cm −1 (C-O-C stretching), 1407 cm −1 (C=C stretching), 1442 cm −1 (coupling of C N and N H), and 996 cm −1 (CO stretching) [55].The detection of carbonate group bands suggests that phosphate groups in hydroxyapatite are partially replaced by carbonate, a phenomenon commonly observed in biological apatites.This replacement can boost the bioactivity of the composite matrix.The change in the carboxylic stretch (CO) frequency from 1641 to 1622 cm −1 suggests the presence of intermolecular interactions between the scaffolds and medicines.The observed shift in peak positions and the emergence of additional peaks after drug loading provide evidence of the successful integration of DOX and CDDP into the composite matrix.The alterations observed in these intermolecular interactions can have an impact on the controlled release features of the matrix, thereby assuring a continuous and precise delivery of the chemotherapeutic drugs.FESEM analysis was used to examine the surface morphology and microstructure of the composite scaffolds (Fig. 5B).The case of a HAp-K-PVA composite scaffold showed the porous inner network structure and the distribution of the constituent materials.The linked porous network of the scaffolds is crucial for bone regeneration since it allows for the migration of drugs and the diffusion of body fluids.The presence of PVA solution, which contains negative side groups on the polymer surface, triggered the creation of pores by interacting with Ca 2+ ions.When HAp is added to a PVA matrix, it was observed that the resulting microstructure has increased porosity aiding in nutrient transport and the formation of extracellular matrix promotes cell adhesion, cell-to-cell communication, and bone repair.The porous architecture of the K-PVA composite matrix plays a pivotal role in promoting bone regeneration and optimizing drug release efficacy [56].Pores are essential for bone tissue formation, enabling migration, vascularization, and proliferation of osteoblast and mesenchymal cells [57].Hulbert et al. found that a minimum pore size of ∼100 μm is needed for mineralized bone regeneration.Larger pores (100-200 μm) facilitate significant bone ingrowth, while smaller pores (10-100 μm) result in fibrous or demineralized osteoid tissue ingrowth [58].Mimicking the natural bone structure, it acts as a scaffold conducive to cell adhesion, proliferation, and differentiation, fostering the formation of new bone.Moreover, it facilitates the controlled incorporation and distribution of therapeutic agents within the surrounding tissue, ensuring targeted and sustained drug delivery.The interconnected pores allow efficient dispersion of pharmaceuticals throughout the matrix enabling precise localization of drug action.
The elemental composition of the cytostatic microbead-loaded HAp-K-PVA composite matrix scaffolds was evaluated using EDS (Fig. 5C).The scaffolds loaded with DOX had a weight proportion of Cl; scaffolds containing CDDP had a weight percentage of Pt; and scaffolds that were co-loaded with both medications had both Cl and Pt as part of their elemental makeup.Ca and O were the two main components found in the sample along with some weight proportions to Si, P, C, and N corresponding to HAp and K present in the composite scaffolds.The scaffolds containing DOX exhibited a detectable Wt.% of chlorine (Cl) which indicates that DOX has been successfully integrated into the composite matrix, since chlorine is a defining ingredient in the chemical composition of doxorubicin hydrochloride.Similarly, the presence of platinum confirms the successful incorporation of cisplatin into the scaffolds, as platinum is a crucial component of cisplatin's molecular structure.The scaffolds co-loaded with both DOX and CDDP, resulted in the presence of both chlorine (Cl) and platinum (Pt) as elements in their composition.This illustrates the practicality of integrating both medications into the composite matrix at the same time, which is crucial for combination chemotherapy approaches that seek to achieve synergistic anticancer effects.Calcium and phosphorous indicate the composite matrix's hydroxyapatite (HAp), which is biocompatible and osteoconductive, ideal for bone regeneration.P, C, and N indicate HAp and keratin's structural and functional synergy in the composite matrix.Phosphorus gives the scaffold strength and durability, while keratin and PVA carbon and nitrogen make it flexible and biodegradable.This combination allows the scaffold to facilitate bone growth and disintegrate slowly, coordinating with tissue repair [59].EDS analysis reveals the composition of HAp-K-PVA composite matrix scaffolds and validates the regulated drug distribution in scaffolds and bone regeneration capability by validating critical components associated with loaded medicines and matrix ingredients.
The average contact angle test result was found to be 51.2°,indicating the hydrophilic nature of the HAp-K-PVA composite scaffold (Fig. 5D).It was observed that as the HAp content of the composite hydrogel is increased; it causes an increase in the surface hydrophilicity.The hydrophilic property of keratin-PVA composite matrix enhances cellular interactions, promoting cell adhesion, distribution, and tissue integration.This property also improves the therapeutic efficacy and bioavailability of hydrophilic drugs such as doxorubicin and cisplatin, by aiding their diffusion and release.Additionally, its biocompatibility minimizes cytotoxicity, promoting favorable cellular responses and tissue regeneration.Due to these properties, co-delivery applications should benefit from enhanced anticancer effects and reduced systemic toxicity.

Microbeads drug loading
Significant variations were seen in the loading capacities of DOX and CDDP, especially with respect to saturation time (Fig. 6A).Around 90% of the original DOX concentration of 60 µg was loaded into β-TCP beads in around 60 min.The loading of the equal quantity of CDDP and DOX co-loaded CDDP took similar time of 36 h.A similar study reported earlier for the loading time matched with the present findings [60].The medication was absorbed more slowly after the first steady and linear loading, which accounted for up to 80% of the total dose.The more loading times for CDDP is attributed to its lower water solubility as compared to the water-soluble DOX.Since the DOX composed of a naphthacenequinone nucleus and daunosamine, an amino sugar, it possesses hydrophilic domains for faster loading [61].DOX easily dissolved in water and added to the K-PVA composite matrix solution during development.However, including CDDP in the K-PVA composite matrix was not feasible due to its limited solubility in waterbased solutions, thus even meager quantities of CDDP (up to 60 μg/ml) could be dissolved in distilled water in agitation.

Microbeads drug release
Distinct drug release behaviors were observed for both cytostatic, DOX, or CDDP, whether loaded in microbeads or incorporated into the HAp-K-PVA composite matrix, individually or co-loaded.During the first 3 days of exposure, the microbeads containing CDDP displayed a rapid and outburst release of about 30% due to the drug present at the surface of scaffolds during its loading.In contrast, samples containing DOX showed a rapid initial release of 20%, after which it showed a uniform drug release.Nearly 48% of the DOX contained in the microbeads was dispersed in Tris-HCl over 30 days.The microbeads which were co-loaded with cytostatic showed a sustained drug release behavior where around 10% CDDP and 15% DOX were released in the first week after which it showed a uniform drug release which is due to the synergistic effect of loading both the cytostatic together (Fig. 6B).

HAp-K-PVA composite matrix drug release
It was observed that DOX dissolves easier and faster in aqueous solution as compared to CDDP when 30 µg/ composite of DOX and CDDP was added to the suspension to integrate them into the HAp-K-PVA composite matrix scaffolds.HAp-K-PVA composite matrix was evaluated, namely R, DM, CM, SM-DB, SM-CB, DM-DB, CM-CB, SM-(C + D)-B, DM-(C + D)-B and CM-(C + D)-B for cytostatic release behavior (Fig. 6C).Composite matrix comprising CDDP-and DOX-loaded microbeads showed similar release behavior as in microbeads, although with a decreased amount of each drug being released.For reference composite scaffolds containing microbeads co-loaded with cytostatic showed a sustained release behavior with around 6% CDDP and 9% DOX release in the first week with sustained drug release till 30 days.A sustained release behavior was observed for CDDP from composite scaffolds incorporating microbeads co-loaded with cytostatic, with around 7% CDDP and 9% DOX release in the first week and subsequent persistent drug release behavior until 30 days.There was a consistent and sustained drug release behavior till 30 days in composite scaffolds including microbeads coloaded with cytostatic, with roughly 6% CDDP and 10% DOX released in the first week.The drug release from the microbeads differs from the release with the HAp-K-PVA composite matrix scaffolds because microbeads are introduced directly into the body, and they sustain the release of the drugs.However, the rationale for incorporating them into the composite matrix (HAp-K-PVA) lies in the composition of the material, which comprises hydroxyapatite and keratin.Hydroxyapatite fills bone defects, while keratin contains cell-binding and adhesion motif binding sequences, facilitating drug diffusion along specific pathways.Due to the porous interconnected structure and adequate pore size in matrix and microbeads, the loaded cytostatic drugs could be released throughout the composite.The HAp-K-PVA composite scaffolds demonstrate a sustained release of CDDP and DOX, correlating well with therapeutic windows by maintaining drug release over 30 days.This controlled release enhances clinical relevance by potentially optimizing therapeutic efficacy.

Antimicrobial studies of DDS
As therapeutic agents, antimicrobial compounds enable targeted medication delivery, minimizing systemic side effects and mitigating the risk of drug resistance.Numerous studies have highlighted the promising antimicrobial properties of HAp-K-PVA composites (Belcarz et al., 2009).It was observed that the composite samples exhibited good antimicrobial activity as there was a noticeable reduction in bacterial growth due to the formation of the inhibition zone.The level of inhibition against S. aureus and Pseudomonas bacterial strains was determined through the antimicrobial test using the test zone of inhibition method.Different composite samples were evaluated, namely R, DM, CM, SM-DB, SM-CB, DM-DB, CM-CB, SM-(C + D)-B, DM-(C + D)-B, and CM-(C + D)-B (Fig. 7A).The sensitivity resistance of bacteria and the diameter of the zone of inhibition are calculated using Eq. 1.
The results demonstrated a distinct zone of inhibition indicating the effective antibacterial properties (1) of scaffolds (Table 3) displaying an inhibition zone of approximately 14.45 mm against S. aureus and 13.33 mm against Pseudomonas.In contrast, the simple matrix with DOX beads configuration exhibited a remarkable increase in the inhibition zone diameter against S. aureus, reaching to 25.52 mm, while remaining at 13.72 mm against Pseudomonas.While certain configurations like DOX matrix and CDDP matrix showed relatively consistent inhibition zones against both bacterial strains, others exhibited notable differences in effectiveness.Notably, under the CDDP matrix with co-loaded DOX and CDDP beads condition, the inhibition zone diameter against S. aureus measured 13.69 mm, whereas it appreciably increased to 21.84 mm against Pseudomonas.In the composite Hap-K-PVA, the antimicrobial properties are primarily attributed to keratin.Keratin acts as an effective barrier against microbial attacks, and reports on microbial degradation of keratin are rare [62].The presence of K in the sample renders the bacterial cells metabolically  inert, ultimately leading to their death and the formation of a clear zone of inhibition on the culture plate.

Swelling rate and degradation studies of DDS
The swelling rate studies were performed for different drug-loaded HAp-K-PVA composite matrices were evaluated.The swelling strength of matrices is a crucial biomaterial parameter for tissue engineering applications, and porosity is an important factor in nutrition transport, metabolite exchange, and body fluid absorption [63].Additionally, swelling causes the pore volume to enlarge, increasing the inner surface area and volume and raising the possibility of cell infusion from the outside to the inside.A larger swelling ratio, however, has a detrimental impact on the scaffold's mechanical stability.
The swelling rate of the scaffold exponentially increases in the first 10 min, with each scaffold showing the following swelling rate R (18%), DM (32%), CM (26%), SM-DB (50%), SM-CB (18%), DM-DB (30%), CM-CB (25%), SM-(C + D)-B (28%), DM-(C + D)-B (25%) and CM-(C + D)-B (45%) after which the swelling gradually increased up 2 h (Fig. 7B).Drug-loaded HAp-K-PVA composite scaffolds exhibited a significant rise in degradation rate during the first week, followed by gradual increase over the next 30 days.With an initial degradation rate of 50%, the CM scaffold displayed the highest rate of material breakdown, potentially compromising the stability of scaffolds.One of the key ideas in bone tissue engineering is the scaffold materials should degrade when being treated for an injury at a pace that is proportional to the rate of bone creation to maintain the rigidity of the bone-scaffold system and permit bone growth [64].If the scaffold experiences a rapid degradation rate, its porous structure may collapse, hindering mass transfer and potentially leading to tissue necrosis.Conversely, a sluggish breakdown rate may result in the formation of fibrous capsules, impeding integration with host tissue and complicating tissue regeneration.Therefore, encouraging tissue regeneration via optimum scaffold breakdown kinetics is crucial [65].The degradation studies performed for different drugloaded HAp-K-PVA matrix composite samples were evaluated over 30 days in Tris-HCl (Fig. 7C).The degradation rate of the scaffolds exponentially increases in the first week, with each scaffold showing the following degradation rates R (42%), DM (40%), and CM (50%) which increased to (15%) in 2-week duration, SM-DB (27%), SM-CB (26%), DM-DB (38%) which increased to 48% in the second week, CM-CB (27%) which increased to 35% in the second week, SM-(C + D)-B (31%) which increased to (45%) in 25 days, DM-(C + D)-B (38%) which increased to (50%) in the second week and CM-(C + D)-B (45%) after which the degradation rate gradually increased up to 30 days.The optimized scaffold degradation kinetics is crucial for effective tissue regeneration.While the gradual degradation may lead to fibrous capsule formation, the rapid degradation risks structural collapse.The DM-(C + D)-B scaffold exhibited a balanced degradation profile, with a 38% increase in the first week and 50% increase in the second week, thereby supporting continuous tissue integration and regeneration.

Cell viability and in vitro analysis
UTOS cell line (osteoblast-like osteosarcoma) cultured in Dulbecco's modified eagle's medium (DMEM F12, Thermo Fisher Scientific) with 5% CO 2 and 10% heatinactivated fetal bovine serum at 37 °C was used to evaluate the biocompatibility and cellular proliferation ability of the HAp-K-PVA composite matrix.The biocompatibility of the microbead-loaded HAp-K-PVA composite matrix was assessed for administration within the human body.By Good Lab Practices and International Organization for Standardization regulations for US Food and Drug Administration approval of drugs and drug delivery vehicles, osteosarcoma cell lines were utilized to perform an MTT assay (Fig. 7D).The analysis of cell viability data for different composite matrix scaffolds indicated a significant decrease in cell viability from the 24-h time point through the first week.The observed results indicate that there was a significant reduction in cell viability in scaffold R. Additionally, it was observed that DM displayed a considerable reduction in cell viability, approximately 67.06% lower than for the R group.Similarly, CM demonstrated a significant decrease in cell viability, reducing approximately 65.89%.Furthermore, the SM-DB scaffold exhibited a substantial decrease in cellular viability, approximately 71.67% lower than for the R group.The observed cell viability of SM-CB showed a substantial decrease, estimated to be around 61.86%.The treatment with DM-DB demonstrated a significant decrease in cell viability, estimated at around 61.42%.The CM-CB, SM-(C + D)-B, DM-(C + D)-B, and CM-(C + D)-B composite matrix scaffolds exhibited a notable decrease in cell viability, with reductions of 65.00%, 63.18%, 73.16%, and 65.87% respectively, indicating the progressive drug release and effect.The cell viability was shown for 24 h, which showed nearly 90% cell viability for HAp-K-PVA composite matrix scaffolds.After 72 h, the cell viability was decreased for all the scaffolds, indicating that the combined synergistic effect of the co-loaded drugs was enhanced.The intracellular metabolism leads to mitochondrial injury and apoptosis after the saturation of metabolic capacity to reduce MTT is achieved.Further, the time-dependent loss of membrane integrity due to formazan exocytosis collectively contributes to the observed decrease in cell viability.Scaffold R showed a significant reduction in cell viability, like DM, CM, SM-DB, and others, demonstrating the effectiveness of the loaded drugs.Furthermore, the synergistic effect of co-loaded drugs is apparent in the more significant reduction in cell viability observed with combined formulations such as SM-(C + D)-B and DM-(C + D)-B.These studies could ultimately translate into improved preclinical outcomes for animal models and contribute to developments in regenerative medicine for clinical test.

Discussion
Microbeads co-loaded with CDDP/DOX were successfully administered into HAp-K-PVA matrix composite scaffolds.The synergistic and combinatorial impact of the two cytostatic toxicity on cancer cells with sustained drug release was investigated in this research over 30 days.The β-TCP microbeads were prepared via droplet extrusion along with ionotropic gelation and were kept in a cross-linking solution of calcium chloride and the release of Ca 2+ ions may compete with other ions for binding sites in alginate, speeding up the cross-linking process.To improve scalability and reproducibility, various optimizations can be further implemented in membrane extrusion, supercritical fluid technology, microfluidics, and preparation methods, including droplet extrusion automation, cross-linking conditions optimization, scalable heating processes, and controlled drug loading techniques [66].A multistep procedure is required to prepare β-TCP microbeads for controlled drug delivery.This assures that a porous structure is appropriate for loading and releasing therapeutic drugs such as doxorubicin and cisplatin.Chemical precipitation is the first stage, in which precursor liquids react to create β-TCP particles.To prevent agglomeration and attain the appropriate particle size and morphology, homogeneous particle formation and reaction condition control are essential.High temperatures, up to 1100 °C, are applied to the precipitated β-TCP to improve its crystallinity and to eliminate the organic remnants.To avoid phase changes impacting the microbead's mechanical characteristics and bioactivity, the temperature profile is kept under control.The drug loading and release kinetics depend on the pore's characteristics such as pore size distribution of developed β-TCP microbeads, ensuring effectiveness of medication delivery.The pore diameter of non-sintered β-TCP beads was analyzed using ImageJ which was found to be between 0.02 and 2043 µm, which is large enough to allow the diffusion of small molecules with sizes of a few nanometers, such as CDDP [67].The porous nature of the microbeads was shown in SEM images, resulting from freeze gelation and drying, which facilitates easy access to the drugs during loading.The composite should function as a drug carrier as well as a scaffold to encourage bone regeneration and this method allows for the fabrication of implants with customized shapes of scaffold as per the requirement of the individual patient [68].Incorporating cytostatic into the microbeads loaded in scaffolds prevents the drug from spreading and migrating uncontrollably in the surrounding tissues resulting in prolonged drug release with target drug delivery.For controlled toxicity via cytostatic, all components used in preparation were non-toxic, with CaP selected owing to its resemblance to a real bone [69] as they leave even more and bigger pores to improve bone ingrowth and supply.There would be no need for further surgery to remove the implant if the beads/matrix composites degraded over time and were replaced with healthy natural bone.It was observed that there were significant variations in saturation time among drug loadings; compared to CDDP, microbeads loaded with DOX released at a faster rate.While chloride ions might impede CDDP binding, their presence remains crucial for the desorption process, thus maintaining the overall uniform loading behavior [70].DOX showed increased release in Tris-HCl buffer which may be due to the carboxyl groups on alginate molecules binding to the amine groups on DOX molecules [71].It may also be inferred that DOX binds poorly to β-TCP [72].When CDDP is hydrolyzed in aqueous solutions, the positively charged species are produced when chloride ions are replaced by water molecules accounting for the adsorption of CDDP on β-TCP through electrostatic interactions.The hydrolysis of CDDP is reversed by chloride ions in the release media, preventing CDDP from adhering to β-TCP once again [73].When the unbound drug volume was rapidly released out of the pores, CDDP showed a rapid initial release over the first three days.DOX exhibited a more sustained release than CDDP.Regardless of whether DOX or CDDP was loaded in microbeads or HAp-K-PVA composite matrix, the release of both medications was drastically reduced in co-loaded matrix composites showing a sustained drug release.In contrast to conventional DDS-like nanoliposomes, which often face burst release and systemic toxicity issues, the composite matrix offers a sustained and localized release of the drugs.By maintaining a constant therapeutic concentration at the tumor site, this controlled release method improves the treatment's effectiveness and safety.Efficient drug levels are sustained for a prolonged duration due to the efficient modulation of DOX and cisplatin release kinetics by the K-PVA composite matrix.The adverse side effects are reduced due to the composite matrix's ability to localize drug delivery to the tumor site, thus minimizing the exposure of healthy tissues to toxic drug concentrations.Biocompatible materials such as hydroxyapatite and PVA prevent inflammatory responses by facilitating improved integration with biological tissues.

Conclusion
The fabrication of microbeads loaded with a composite matrix for osteosarcoma, along with the synthesis and characterization of K from human hair and HAp from eggshells, has yielded promising results with potential applications in cancer medicine.The use of natural resources in the production of K and HAp is both environmentally friendly and cost-effective.The incorporation of DOX and CDDP into microbeads and the HAp-K-PVA composite matrix was confirmed by physicochemical and thermal characterization.Moreover, compared to the drug release from DOX-loaded beads alone, the composite matrix exhibited a significantly reduced release rate of approximately 48% over 30 days.In contrast to CDDP, which rapidly releases its contents within 3 days regardless of its formulation, DOX exhibits a sustained release profile over 30 days.This sustained release, combined with the persistent cytostatic effect of CDDP and their synergistic action, makes co-loaded microbeads/matrix scaffolds highly effective for treating osteosarcoma.Following biodegradation and swelling tests, these microbead-loaded HAp-K-PVA scaffolds exhibited promising antibacterial activity and cell viability against the UTOS cell line.However, further in-depth in vivo investigations are still required to evaluate their performance, biocompatibility, and long-term effects before clinical implementation.Additional research is needed to investigate factors including drug metabolism, immune response, and the actual interactions of the tumor microenvironment.The efficacy and durability of the K-PVA composite matrix in biological environments over the long term remain unknown.To evaluate the sustained efficacy and durability of the drug delivery system, further extended in vivo studies are necessary to completely actualize and authenticate the potential of the K-PVA composite matrix as a controlled release mechanism for chemotherapeutic agents.The presented research focused primarily on in vitro investigations, which do not fully match the complexity of in vivo settings, and thus, further investigation in this direction is a prerequisite.However, this study established a promising foundation for novel and sustainable biomaterials for osteosarcoma treatment.Scaling up with in vivo and preclinical investigations could provide a more comprehensive understanding of the implications and translate into enhanced clinical outcomes.This advancement could ultimately translate into improved clinical outcomes for patients and contribute to developments in regenerative medicine.

Fig. 1 A
Fig. 1 A Synthesis of HAp from waste-derived eggshells; B K extraction process from discarded human hair; C Schematic representation of the preparation of microbeads; D Schematic diagram of HAp-K-PVA composite matrix scaffolds

2 .
75 cm −1 , indicating the existence of a C-O bond, and the strongest peaks at 3641.24 cm −1 attributed to O-H stretching vibrations, indicating the formation of CaO and Ca(OH) 2 .After undergoing chemical precipitation and subsequent calcination, the elimination of C-O and O-H bonds in the samples occurred.This transformation was attributed to the conversion of CaO and Ca(OH) 2 molecules into HAp, leading to a shift in the peak to CO 3 −The most prominent peak in all the samples was located at 1028 cm −1 , and it related to the v3 antisymmetric stretch vibrations of the PO 4 3− bonds.All the samples include evidence of the v1 symmetric stretch vibrations of PO 4 3−

Fig. 2 A
Fig. 2 A XRD Plot of HAp; B FTIR Spectra of HAp; C HR-TEM Analysis of HAp; D SEM Image of HAp; E EDS Spectra of HAp; F Particle size of HAp; G Zeta potential of HAp

Fig. 3 A
Fig. 3 A FTIR spectra of K; B XRD Plot of K; C UV Spectrophotometer analysis of K; D Lowry estimation of K

Fig. 6 A
Fig. 6 A DOX and CDDP loading of TCP beads as a proportion of the initial drug concentration (60 µg/ml) versus the saturation period.Both cytostatic were dissolved in water for injection (WFI); B Drug release from microbeads DOX-, CDDP-and co-loaded in Tris-HCl over 30 days; C Drug release from HAp-K-PVA composite scaffolds DOX-, CDDP-and co-loaded in Tris-HCl over 30 days

Fig. 7 A
Fig. 7 A Swelling rate of HAp-K-PVA composite scaffold; B Antimicrobial test on HAp-K against Staphylococcus aureus (S. aureus) and Pseudomonas species; C Degradation rate of HAp-K-PVA composite scaffolds; D MTT Assay of HAp-K-PVA composite scaffolds showing cell viability at 24 h and 72 h

Table 1
Different HAp-K-PVA composite matrix scaffolds considered in this study with different cytostatic concentration

Table 2
Elemental composition of microbeads with Ca/P ratio

Table 3
Zone of inhibition of antimicrobial test on HAp-K-PVA scaffolds against Staphylococcus aureus (S. aureus) andPseudomonas species