The Combined Anti-Tumor Efficacy of Bioactive Hydroxyapatite Nanoparticles Loaded with Altretamine

In the current study, the combined anti-tumor efficacy of bioactive hydroxyapatite nano- particles (HA-NPs) loaded with altretamine (ALT) was evaluated. The well-known fact that HA has great biological compatibility was confirmed through the findings of the hemolytic experiments and a maximum IC50 value seen in the MTT testing. The preparation of HA-NPs was performed using the chemical precipitation process. An in vitro release investigation was conducted, and the results demonstrated the sustained drug release of the altretamine-loaded hydroxyapatite nanoparticles (ALT-HA-NPs). Studies using the JURKAT E6.1 cell lines MTT assay, and cell uptake, as well as in vivo pharmacokinetic tests using Wistar rats demonstrated that the ALT-HA-NPs were easily absorbed by the cells. A putative synergism between the action of the Ca2+ ions and the anticancer drug obtained from the carrier was indicated by the fact that the ALT-HA-NPs displayed cytotoxicity comparable to the free ALT at 1/10th of the ALT concentration. It has been suggested that a rise in intracellular Ca2+ ions causes cells to undergo apoptosis. Ehrlich’s ascites model in Balb/c mice showed comparable synergistic efficacy in a tumor regression trial. While the ALT-HA-NPs were able to shrink the tumor size by six times, the free ALT was only able to reduce the tumor volume by half.


Introduction
Apoptosis is a fundamental biological process that multicellular organisms use to replace aging, damaged, or unhealthy cells. Caspases (protein-digesting enzymes) are activated by an extrinsic or intrinsic mechanism, initiating a tightly controlled process that ultimately results in cell death. TNF-α, and Fas are examples of extracellular ligands that begin the extrinsic pathway, whereas stress-induced intracellular signals start the intrinsic pathway [1]. One of the mechanisms that stimulate intracellular apoptotic signaling is increased intracellular calcium ion (Ca 2+ ) concentrations. Cancerous cells are currently being treated with chemotherapy employing substances that interfere with the calcium ion homeostasis in cells. Kim et al. examined the effects of panaxydol, which was extracted from Panax Ginseng roots, on MCF-7 (human breast cancer) cells. They discovered that the substance might cause cell death and raise intracellular Ca 2+ ion concentrations [2]. Nagoor Meeran et al. investigated the effects of carvacrol, a phenol-derived (monoterpenoid) from cytoplasm. In other words, it is believed that the presence of calcium content activates the Calpain, as Ca 2+ -dependent protein kinases disrupt the cytoskeleton. This results in the disruption of the cytoskeleton around HA-NPs. Additionally, the high surface energy of HA-NPs due to the abundant Ca 2+ ions can stimulate certain molecules and create free radicals. Actin filament damage caused by ROS can compromise the integrity of the cytoskeleton and cause cytoskeleton disruption [12]. Ca 2+ can also activate NO synthases, transglutaminase, calcineurin, endonucleases, or phospholipases. These substances have been demonstrated to play a role in the different types of apoptotic cell death. The role of calcium-binding proteins including ALG-2 or calcium/calmodulin-dependent kinases such as the death-associated protein kinase and other enzymes known to be vital in apoptosis are only a few examples of other pathways that have been reported [13].

Materials
Altretamine (ALT) was received from Spectra Lab. Hyderabad, India, as a gift sample. HiMedia Lab Pvt. Ltd., Mumbai, India provided the calcium nitrate tetrahydrate (Ca(NO 3 ) 2 .4H 2 O), ammonium dihydrogen phosphate (NH 4 H 2 PO 4 ), ammonia solution, methanol, and isopropyl alcohol that was needed. HPLC-grade water, methanol, and acetonitrile were purchased from Sigma-Aldrich, Bangalore, India. Internally prepared Milli-Q water was used during the trials. There were also analytical-grade reagents employed.

Synthesis ALT-Loaded HA-NPs (ALT-HA-NPs)
HA-NPs were prepared by using the wet chemical precipitation approach [14]. Firstly, separate aqueous solutions of 0.025 M (Ca(NO 3 ) 2 and 0.025 M of (NH 4 ) 3 PO 4 were made using Milli-Q water. After that, the previously prepared Ca(NO 3 ) 2 solution in 500 mL of the three-necked round-bottom flask with a rosary-type condenser in the center neck was placed in a silicon oil thermal bath and kept at 90 • C under magnetic stirring conditions [15]. An aqueous (NH 4 ) 3 PO 4 solution and NH 4 OH solution were added dropwise via the left neck of the flask, and the pH was determined using a potentiometer mounted on the right neck of the flask. To produce HA particles, the solution was allowed to stir for 3 h until the required pH level was obtained [16]. Equation (1) depicts the chemical precipitation reaction for the production of HA. ALT was physically adsorbed onto the prepared HA-NPs. 6(NH 4 ) 2 HPO 4 + 10Ca(NO 3 ) 2 ·4H 2 O + 8NH 4 OH → 20NH 4 NO 3 +Ca 10 (PO 4 ) 6 (OH) 2 + 46H 2 O (1)

Determination of the Encapsulation Efficiency % and Loading Capacity % of ALT-HA-NPs
The encapsulation efficiency % (EE %) of ALT in the prepared HA-NPs was determined by an indirect method where 2 mL of the prepared nanoparticle dispersion was centrifuged at 15,000 pm for 45 min. The supernatant was separated and analyzed spectrophotometrically at 226 nm after appropriate dilution to determine the free ALT [17]. The encapsulation efficiency was calculated using Equation (2): Additionally, the drug loading capacity (DL%) was calculated [18] using Equation (3): (DL%) = The amount of loaded ALT in nanoparticles (mg) weight of nanoparticles (mg) × 100 (3)

Particle Size Analysis of ALT-HA-NPs
The particle size of the prepared ALT-HA-NPs was determined by dynamic light scattering technique using zetasizer (Malvern Instruments Ltd., Malvern, UK). The sample was diluted to the appropriate concentration, and the measurements were carried out at a temperature of 25 • C and an angle of 90 • [19]. The surface morphology was determined by TEM (JTEM model 1010, JEOL ® , Tokyo, Japan). One drop of the prepared ALT-HA-NPs was added to the collodion-coated copper grid after being diluted with distilled water. The dried sample was stained with uranyl acetate solution and examined with TEM [20].

Scanning Electron Microscopy (SEM)
A small amount of the dry powder of the prepared ALT-HA-NPs was coated with approximately 15 nm gold (SPI-Module Sputter Coater). The coated sample was scanned by an analytical scanning electron microscope (JSEM-6360LA, JEOL ® , Tokyo, Japan) under vacuum conditions at 15 kV acceleration voltage at room temperature [21].

In Vitro Release Studies
The drug release study was performed using the dialysis method. Where 2 mL (3 mg equivalent of ALT) of the prepared ALT-HA-NPs was transferred into a dialysis bag (cutoff 12kDa, Sigma). The dialysis bag was immersed in a beaker containing 100 mL of Phosphate Buffer Saline (PBS; pH 7.4) as a dissolution medium. The dissolution medium was stirred at 100 rpm and kept at 37 ± 1 • C. The samples were withdrawn at different time intervals up to 24 h and analyzed spectrophotometrically at 226 nm after appropriate dilution. The experiment was performed in comparison with the free ALT in triplicate and the mean ± SD was calculated [22,23].

Hemolytic Activity Determination
A previously reported procedure was used to evaluate the hemolytic activity of whole blood. Briefly, heparinized blood was obtained from dependable sources (Research Ethics Review Board (ERB) approval no. 2022/20137/MedAll/TRY; dated 11-02-2022). Before the experiment, human red blood cells were twice washed in PBS (pH 7.4). In 96-well microtiter plates, 100 µL of human RBC was plated with 0.4% (v/v) in PBS suspension and subsequently incubated for up to 24 h at 37 • C with an equal amount of ALT, ALT-HP-NPs, normal saline (0% hemolysis) aided as the negative control, and Milli-Q water (100% hemolysis) aided as the positive control. Equation (4) is the formula used to compute the % hemolysis in the experiment, which was done in triplicate [24]. In an ELISA reader, the lysed RBC absorbance was measured at 540 nm. PBS and 1% Triton-X 100 were used to determine 0% and 100% hemolysis.

In Vitro Cell Line Studies Cytotoxicity Assessment
A previously described procedure was slightly modified to investigate the ALT, HA-NP, and ALT-HA-NP cytotoxicity. The MTT test on JURKAT cells was used to measure the percentage of cytotoxicity (E6-1; acute T cell leukemia cells). Briefly, the cells were seeded in a 96-well microtiter plate at a mass of 5 × 10 3 cells·well −1 , supplemented with fetal calf serum (2.5%), and then incubated at 37 • C for 24 h with a N 2 atmosphere of 90% with 5% of CO 2 , and 5% O 2 for 24, 48, and 72 h, respectively. The cells were treated with two concentrations of the samples, i.e., 150 µg·mL −1 , and 300 µg·mL −1 . For this addition of 20 µL of MTT solution (5 mg·mL −1 in PBS at pH 7.4) to each well subsequently, the required amount of time had passed. Then, DMSO (100 µL) was used to dissolve the formazan crystals [25]. At 540 nm, the optical density (OD) was determined from an ELISA microplate reader after mixing with a mechanical plate mixer (Synergy HT, BioTek, Winooski, VT, USA). Three copies of each measurement were made. After 24 h, IC 50 values were collected for each sample, and the following equation (Equation (5)) was used to calculate the percentage of cytotoxicity: 2.9. In Vivo Studies 2.9.1. Animals The pharmacokinetic experiments were performed on male Wistar rats. The Institutional Animal Ethics Committee (IAEC), Anna University, Chennai, India approved the study protocol (Approval No. 1338). The animals were purchased from DORENCAMP, Bharathidasan University Animal House Capacity (IAEC/2021-2022; dated 28 August 2021). Utilizing Ehrlich's ascites model on Balb/c mice, tumor regression activity was assessed. The animals were kept in an animal house that was registered with CPCSEA (2276/RO/24/p/CPCSEA). IAEC authorized the protocol, which has the protocol number IN/TN/IAEC/2018/07. Animals were kept in normal laboratory cages made of polypropylene, two to a cage with unrestricted access to a normal laboratory diet and water (ambient temperatures of 25 ± 2 • C and 55 ± 5% relative humidity).

Pharmacokinetic Evaluation
Each of the two groups of animals contained six total individuals. One group received the ALT-HA-NPs (dispersed in PBS) for the studies, whereas the second group received the pure ALT (dispersed in PBS). Half a milliliter of the dispersions was administrated through the tail vein by intravenous administration. Each animal was given 0.2 mL diethyl ether anesthetic before the blood samples were taken after the retro-orbital plexus and immediately preserved within EDTA vacationers. The blood was withdrawn at the subsequent intervals: 0, 5, 15, 30, and 45 min, and 1, 2, 4, 6, 12, and 24 h. The bloodcontaining samples were then centrifuged at 4 • C for 10 min using a 5000-rpm machine (Eppendorf). Before further estimation, in Eppendorf tubes, the supernatant was collected and kept at −20 • C. The analysis method used by HPLC was modified from that used by Mohamed et al. [26]. The C max , T max , AUC (0-t) , t 1/2 , and the elimination rate constant was computed using the Microsoft Excel Addins-PK Solver and Pk1Pk2. To calculate relative bioavailability (Bio rel ), Equation (6) was used: where, AUC-Area under the Curve; D-dose.

Tumor Regression Evaluation
The Ehrlich's ascites (EAC) model in Balb/c mice was used to test the in vivo anticancer activity. Subcutaneously, the EAC cells (100 µL) were injected into the Balb/shaved dorsal surface. Seven days after the inoculum, measurable tumors formed (0.08 cm 3 ). The mice were separated into four groups (n = 6) after the initial tumor development. PBS was injected into the first group as a control, ALT-HA-NPs was injected into the second group once every two days (1.5 mg/200 µL), free ALT was injected into the third group once in two days/mice (129 µg/200 µL), and HA-NPs were injected into the fourth group once every two days (1.5 mg/200 µL). Each group received intravenous care through a tail vein. Every other day, vernier calipers were used to measure the tumor length and width, and Equation (7) was used to compute the tumor volume [27].
The anti-tumor activity was then assessed in each case after measuring the tumor volume up to the 21st day.

Statistical Analysis
The mean and standard deviation of the data are the results of statistical analysis (mean ± SD). GraphPad (2018) Prism and Microsoft Excel (2019) were used to create bar graphs. ANOVA test was used to assess the significant differences among various experimental groups.

Results and Discussion
The most popular method for the preparation of HA-NPs was wet chemical precipitation from a solution, which has several benefits including a straightforward processing route, a high yield, being appropriate for industrial manufacturing, using cheap reagents, and the ability to create products with variable phase composition.
The prepared ALT-HA-NPs exhibited high EE% (96.27 ± 6.78%) and had drug loading of 8.2%. The particle size of the prepared ALT-HA-NPs was small in the nano range (156 ± 10.86 nm). The surface morphology of the prepared ALT-HA-NPs was examined by TEM and SEM. The prepared nanoparticle appeared spherical, as shown in Figure 1. Similar results were obtained by Ansari et al., who developed hydroxyapatite nanoparticles containing epirubicin for the treatment of cancer and examined the prepared nanoparticles with TEM [20]. Additionally, Soleimani et al. prepared Hydroxyapatite Nanoparticles for a human breast adenocarcinoma cell line and fibroblast examined the morphology with SEM and found that the prepared hydroxyapatite nanoparticles appeared spherical [28].
Every other day, vernier calipers were used to measure the tumor length and width, and Equation (7) was used to compute the tumor volume [27].
Tumor volume (TV) = 0.5 × Length × Width The anti-tumor activity was then assessed in each case after measuring the tumor volume up to the 21st day.

Statistical Analysis
The mean and standard deviation of the data are the results of statistical analysis (mean ± SD). GraphPad (2018) Prism and Microsoft Excel (2019) were used to create bar graphs. ANOVA test was used to assess the significant differences among various experimental groups.

Results and Discussion
The most popular method for the preparation of HA-NPs was wet chemical precipitation from a solution, which has several benefits including a straightforward processing route, a high yield, being appropriate for industrial manufacturing, using cheap reagents, and the ability to create products with variable phase composition.
The prepared ALT-HA-NPs exhibited high EE% (96.27 ± 6.78%) and had drug loading of 8.2%. The particle size of the prepared ALT-HA-NPs was small in the nano range (156 ± 10.86 nm). The surface morphology of the prepared ALT-HA-NPs was examined by TEM and SEM. The prepared nanoparticle appeared spherical, as shown in Figure 1. Similar results were obtained by Ansari et al., who developed hydroxyapatite nanoparticles containing epirubicin for the treatment of cancer and examined the prepared nanoparticles with TEM [20]. Additionally, Soleimani et al. prepared Hydroxyapatite Nanoparticles for a human breast adenocarcinoma cell line and fibroblast examined the morphology with SEM and found that the prepared hydroxyapatite nanoparticles appeared spherical [28].

In Vitro Release Outcomes
The ALT in vitro release profile revealed a 24 h cumulative percentage of drug release up to 92.14 ± 4.45%. The ALT-HA-NPs, on the other hand, displayed a biphasic drug release profile, through an initial burst release up to 22.6 ± 1.2% in the initial 3 h, tailed by a continuous release (Figure 2). This first burst release of the ALT may be attributed to the

In Vitro Release Outcomes
The ALT in vitro release profile revealed a 24 h cumulative percentage of drug release up to 92.14 ± 4.45%. The ALT-HA-NPs, on the other hand, displayed a biphasic drug release profile, through an initial burst release up to 22.6 ± 1.2% in the initial 3 h, tailed by a continuous release (Figure 2). This first burst release of the ALT may be attributed to the drug molecules adhering to the nanoparticle surface [29]. The initial quick release may also be attributed to the nanoparticles' great surface-to-volume ratio [30]. The drug is confined inside the orifices of the porous nanoparticles, which results in the second phase of slower sustained release. In 24 h, as much as 71.2 ± 2.1% of the drug was released. An extended length of time will be favored by the therapeutic impact of gratitude to such a slow and steady drug release, which has been documented by Kumar et al. (2015) [31]. Similar results were obtained by Cai et al. who developed zoledronic acid-functionalized hydroxyapatiteloaded polymeric nanoparticles for the treatment of osteoporosis and observed that the drug release displayed a biphasic drug release profile [29].
fined inside the orifices of the porous nanoparticles, which results in the second phase of slower sustained release. In 24 h, as much as 71.2 ± 2.1% of the drug was released. An extended length of time will be favored by the therapeutic impact of gratitude to such a slow and steady drug release, which has been documented by Kumar et al. (2015) [31]. Similar results were obtained by Cai et al. who developed zoledronic acid-functionalized hydroxyapatite-loaded polymeric nanoparticles for the treatment of osteoporosis and observed that the drug release displayed a biphasic drug release profile [29].

Hemolysis Outcomes
To evaluate the NP biocompatibility and demonstrate their safety as a brand-new ALT carrier that satisfies preclinical requirements, a toxicity investigation was carried out. Wide-ranging hemolysis was seen in the case of Milli-Q water; however, isotonic saline was found to be minor. The ALT-HA-NPs had a hemolytic activity of 1.06 ± 0.05% and the unloaded HA-NPs had a hemolytic activity of 0.98 ± 0.04%, respectively ( Figure 3). As a result, the NPs show no detectable hemolytic incompatibility (p < 0.05).

Hemolysis Outcomes
To evaluate the NP biocompatibility and demonstrate their safety as a brand-new ALT carrier that satisfies preclinical requirements, a toxicity investigation was carried out. Wide-ranging hemolysis was seen in the case of Milli-Q water; however, isotonic saline was found to be minor. The ALT-HA-NPs had a hemolytic activity of 1.06 ± 0.05% and the unloaded HA-NPs had a hemolytic activity of 0.98 ± 0.04%, respectively ( Figure 3). As a result, the NPs show no detectable hemolytic incompatibility (p < 0.05).

Pharmacokinetic Parameters
Pharmacokinetic investigations were carried out, and many parameters were evaluated using a non-compartmental study to support our findings. Figure 5 shows the drug levels detected in blood for the mixture of lyophilized drug concentrate and ALT-HA-NPs. Calculations were made based on the data to determine Tmax, Cmax, AUC(0-t), drug half-life, elimination rate constant, and relative (Biorel) bioavailability.
For the free ALT and the ALT-HA-NPs, the Cmax was discovered to be 6124.4 ± 128.34 ng/mL and 1724.73 ± 98.46 ng/mL, respectively (p < 0.001). The Tmax was determined to be 0.5 ± 0.24 h for the free ALT and 4 ± 0.31 h for the ALT-HA-NPs. The ALTs' AUC in solution was determined to be 8243.11 ± 6457.51 ng. h/mL, but when it was loaded into HA-NPs, it was much higher at 19,998.71 ± 8653.421 ng·h/mL (p < 0.001), increasing the ALTs' bioavailability by about 2.5 times. Drug-loaded HA-NPs were shown to have a significantly lower elimination rate constant (0.08756 ± 0.00279) than the free ALT (0.924567 ± The results of the hemolytic study, the cytotoxicity experiment (MTT assay), and a further identically high IC 50 value confirm the spherical-shaped HA-NPs exceptional biocompatibility and non-antigenicity, which were previously described in the literature [32,33]. When HA-NPs dissolve and release calcium ions into the cytoplasm after entering the cancer cell, it is believed that this disturbs intracellular calcium homeostasis. Calcium homeostasis is crucial because calcium plays a vital function as a secondary messenger, regulating crucial cellular processes including apoptosis and cell proliferation [34].

Pharmacokinetic Parameters
Pharmacokinetic investigations were carried out, and many parameters were evaluated using a non-compartmental study to support our findings. Figure 5 shows the drug levels detected in blood for the mixture of lyophilized drug concentrate and ALT-HA-NPs. Calculations were made based on the data to determine T max , C max , AUC (0-t) , drug half-life, elimination rate constant, and relative (Bio rel ) bioavailability.

Tumor Regression Studies
Experiments on the 21-day tumor regression in Balb/c mice used as Ehrlich's ascites model were effective. As expected, the tumor volume was shown to significantly grow in the control group; after 21 days, an average tumor volume of 3221.45 mm 3 was documented. However, the tumor volume was significantly reduced in the other three groups ( Figure 6). The ALT-HA-NPs produced the greatest reduction in tumor volume, with an average end volume of 506.25 mm 3 (p < 0.001). However, the average final tumor volume in the free ALT was reported to be 1151.05 mm 3 (p < 0.01). Furthermore, it was discovered that the blank HA-NPs significantly the reduced tumor size (1782.03 mm 3 ). Overall, the tumor decreased to 1/6th of its volume after being treated with ALT-HA-NPs, compared to 1/2 of its volume when treated with the free ALT and blank HA-NPs ( Figure 6). The cellular uptake study thoroughly demonstrated that the nanoparticles can be readily internalized by the cells, and these findings further support the possibility of a synergistic interaction between the anticancer ALT activity, and the calcium ions released from the carrier, which would result in greater tumor volume reduction than with the anticancer drug alone or with blank HA-NPs [36]. Additionally, it was hypothesized that the drugloaded NPs enhanced the permeation and retention mechanism, which causes them to collect and localize in the tumor area, where they reduce the tumor volume. For the free ALT and the ALT-HA-NPs, the C max was discovered to be 6124.4 ± 128.34 ng/mL and 1724.73 ± 98.46 ng/mL, respectively (p < 0.001). The T max was determined to be 0.5 ± 0.24 h for the free ALT and 4 ± 0.31 h for the ALT-HA-NPs. The ALTs' AUC in solution was determined to be 8243.11 ± 6457.51 ng. h/mL, but when it was loaded into HA-NPs, it was much higher at 19,998.71 ± 8653.421 ng·h/mL (p < 0.001), increasing the ALTs' bioavailability by about 2.5 times. Drug-loaded HA-NPs were shown to have a significantly lower elimination rate constant (0.08756 ± 0.00279) than the free ALT (0.924567 ± 0.023456), and their half-life was found to be higher at 3.2 ± 0.29 h compared to the free ALTs of 0.87 ± 0.22 h.
ALT was thought to be rapidly eliminated from the body and to have a short half-life, which was well supported by Weber et al. (2015) [35]. As a result, it is clear from comparing the kinetic profiles of the two situations that the ALT was released from nanoparticles in a sustained manner and stays in the body for long time. The HA-NPs maintenance of constant plasma drug levels supports the increased T 1/2 values. Comparing the AUC (0-t) values with the extended T max values made it clear that the body was exposed to the drug for a longer amount of time in the case of the drug-loaded HA-NPs. Enhancing bioavailability has advantages. Therefore, the HA nanoparticles of ALT are the best choice for safer therapy with increased drug bioavailability. To make it clinically feasible, further safety and efficacy studies are needed.

Tumor Regression Studies
Experiments on the 21-day tumor regression in Balb/c mice used as Ehrlich's ascites model were effective. As expected, the tumor volume was shown to significantly grow in the control group; after 21 days, an average tumor volume of 3221.45 mm 3 was documented. However, the tumor volume was significantly reduced in the other three groups ( Figure 6). The ALT-HA-NPs produced the greatest reduction in tumor volume, with an average end volume of 506.25 mm 3 (p < 0.001). However, the average final tumor volume in the free ALT was reported to be 1151.05 mm 3 (p < 0.01). Furthermore, it was discovered that the blank HA-NPs significantly the reduced tumor size (1782.03 mm 3 ). Overall, the tumor decreased to 1/6th of its volume after being treated with ALT-HA-NPs, compared to 1/2 of its volume when treated with the free ALT and blank HA-NPs ( Figure 6). The cellular uptake study thoroughly demonstrated that the nanoparticles can be readily internalized by the cells, and these findings further support the possibility of a synergistic interaction between the anticancer ALT activity, and the calcium ions released from the carrier, which would result in greater tumor volume reduction than with the anticancer drug alone or with blank HA-NPs [36]. Additionally, it was hypothesized that the drug-loaded NPs enhanced the permeation and retention mechanism, which causes them to collect and localize in the tumor area, where they reduce the tumor volume.

Tumor Regression Studies
Experiments on the 21-day tumor regression in Balb/c mice used as Ehrlich's ascites model were effective. As expected, the tumor volume was shown to significantly grow in the control group; after 21 days, an average tumor volume of 3221.45 mm 3 was documented. However, the tumor volume was significantly reduced in the other three groups ( Figure 6). The ALT-HA-NPs produced the greatest reduction in tumor volume, with an average end volume of 506.25 mm 3 (p < 0.001). However, the average final tumor volume in the free ALT was reported to be 1151.05 mm 3 (p < 0.01). Furthermore, it was discovered that the blank HA-NPs significantly the reduced tumor size (1782.03 mm 3 ). Overall, the tumor decreased to 1/6th of its volume after being treated with ALT-HA-NPs, compared to 1/2 of its volume when treated with the free ALT and blank HA-NPs ( Figure 6). The cellular uptake study thoroughly demonstrated that the nanoparticles can be readily internalized by the cells, and these findings further support the possibility of a synergistic interaction between the anticancer ALT activity, and the calcium ions released from the carrier, which would result in greater tumor volume reduction than with the anticancer drug alone or with blank HA-NPs [36]. Additionally, it was hypothesized that the drugloaded NPs enhanced the permeation and retention mechanism, which causes them to collect and localize in the tumor area, where they reduce the tumor volume. Figure 6. Anti-tumor activity of free ALT, ALT-HA-NPs, and blank HA-NPs (mean ± SD, n = 3). * significant compared to Control (PBS) group at p ˂ 0.05, ** at p less than 0.01, *** at p less than 0.001 Figure 6. Anti-tumor activity of free ALT, ALT-HA-NPs, and blank HA-NPs (mean ± SD, n = 3). * significant compared to Control (PBS) group at p < 0.05, ** at p less than 0.01, *** at p less than 0.001.

Conclusions
According to our investigations, the ALT-HA-NPs exhibit superior combined anticancer effects compared to the free drug and blank HA-NPs. Due to its polar surface, HA with nanoscale dimensions is ideally adapted to interact with biomolecules in tissues and cells as well as with drugs. The cells may quickly ingest HA-NPs, and as they degrade, more Ca 2+ eventually finds its way inside the cells. Altretamine anti-tumor action is then enhanced by calcium ion-induced proapoptotic pathways. It is necessary to conduct more research in this area to learn more about this synergism and its possible applications. Our investigation clearly shows that the formulation prepared can be used to treat CLL very successfully, minimizing the side effects such as diarrhea, weakness, nausea, and vomiting. For the research to advance, it is more important than ever to have a thorough understanding of the biomolecular effects of HA-NP-based systems on various cancer types. The experimental work undertaken for this purpose is quite complex, requiring the cooperation of biomolecular chemists, biomaterials scientists, oncologists, and biologists, in addition to the evaluation of full in vitro/in vivo toxicity profiles.