Precise Engineering of Cisplatin Prodrug Into Supramolecular Nanoparticles: Enhanced on in Vitro Antiproliferative Activity and Treatment and Care of in Vivo Renal Injury

Renal cell carcinoma (RCC) is a widespread type of urological tumor that derives from the highly heterogeneous epithelium of the kidney tissue. For the past decade, the treatment of kidney cancer cells has changed clinical care for RCC. Herein, we present a very easy and cost-effective method that incorporates tumor-specic targeting supramolecular nanoassembly, and therapeutically to overcome the different challenges raised by the distribution of the pharmaceutical potential anticancer drug Cisplatin (CIS-PT). On covalent conjugations of hydrophobic linoleic acid by carboxylic group, the CIS-PT prodrugs were skilled in impulsively nanoassembly into extremely steady nanoparticles size (~100 nm). Electron microscopic techniques have veried the newly synthesized morphology of CIS-PT-NPs. The anticancer properties of CIS-PT and CIS-PT-NPs against Caki-1 and A-498 (renal carcinoma) cancer cell lines have been evaluated after successful synthesis. Other research, such as dual staining acridine orange/ethidium bromide, Hoechst 33344 and ow cytometry study on the apoptosis mechanisms, have shown that proliferation in renal cancer cells is associated with apoptosis. Further the In vivo toxicity results displays the CIS-PT-NPs remarkably alleviated the toxicity of the potential anticancer drug CIS-PT In vivo while conserving the Pharmaceutical activity. Compared to CIS-PT, CIS-PT-NPs demonstrate excellent In vitro and In vivo property, this study claried the CIS-PT-NPs as a healthy and positive RCC care chemotherapeutics technique and deserve further clinical evaluations. 5. The In vivo toxicity results displays the CIS-PT-NPs remarkably alleviated the toxicity compared to CIS-PT.


Introduction
Renal cell carcinoma (RCC) is not a single organism but rather a group of tumors that arise from the highly heterogeneous epithelium of kidney tissue [1][2][3][4][5]. According to the Heidelberg description of renal cell cancer, histopathological subtypes of RCC includes clear cell adenocarcinoma, the most prevalent form of RCC, chromophobe accumulating duct carcinoma, papillary carcinoma, and unclassi ed carcinoma [6][7][8][9][10]. Among urological tumors, RCC has the highest risk of cancer-speci c mortality and the 5-year survival rate for patients with metastatic disease is just 12%. In comparison, RCC reports for about 3.8% of all adult human cancers, with an average rise in incidence. Cancer treatment is one of the key methods of treating cancer; however, its usefulness is hampered by drug resistance [11][12][13][14].
Cisplatin is a potent chemotherapy agent that has been commonly used as a standard drug for the treatment of different forms of cancer. However, the therapeutic application of Cisplatin is consistent with dose-limiting toxicity (e.g., nephrotoxicity and myelosuppression) [15][16][17][18]. In addition, the endogenous or acquired opioid resistance often greatly impedes its therapeutic bene ts. Numerous formulations, including liposomes, polymer mice and albumin, have been explored to completely exploit platinumbased agents. However, owing to the major formulation problems presented by platinum products, current methods are subject to hurdles such as the need for a vast quantity of excipients and complex production processes [19][20][21]. It is also not concerning that very few nano-medicines have reached clinical trials, although a signi cant number of platinum-encapsulated nano-devices have been described to date. As described above, the formulation of novel platinum-encapsulated candidates reconstructed with simple structural modi cations and self-assembly into supramolecular nanostructures is highly desirable. These activities will advance the therapeutic use of platinum complexes [22][23][24][25][26].
In addition, unperformed modi cation of CIS-PT to inactive metabolites correspondents may be signi cantly inhibited after systemic control due to reduced metabolic properties of in the plasma membranes [27][28][29]. Therefore, the CIS-PT encapsulation prodrug was shown to reduce drugs stimulations and increase the performance of drug loading e cacy by the effects of permeability and retention of enhancement. In this report, we de ned simple and superior method of effect that energetically combine with the supramolecular nanoassembly tumor targeting and drug reconstruction therapies of CIS-PT. To effectively targets the objective, hydrophobic linoleic acid (LA) is an integral fatty acids, by carboxylic group formation, was conjugated with the amino moiety of the CIS-PT equivalent.
Fruitfully, we discovered in the water solutions the CIS-PT prodrugs of increasing self-modeling into the nanoparticles of nanoassembly. The In vitro proliferation of renal cancer cells was investigated by the nanoassembly CIS-PT prodrug and the morphological deviations and cell death examinations of renal cancer cells were identi ed.

Fabrication of CIS-PT NPs
DIEA was combined with CIS-PT (75 mg, 0.20 mol) and hydrophobic linoleic acid (78 mg, 0.20 mol) solutions containing 2 mL of DMF (103 mg, 8.1 mol). The reaction mixture solutions was allowable to stir overnight at 37 o C and then to evaporate to extract the DMF solution. The reaction mixtures that have been absorbed in the DCM. Five percent citric acid, NaCl and aqueous NaHCO 3 is used to wash the organic coating. The DCM solution were removed using vacuum dried with Na 2 SO 4 solution. Figure 1 demonstrates the comprehensive fabrication techniques.

Characterization
The morphology of CIS-PT NPs was tested by transmission electron microscopy (TEM; JEM-1200EX, Japan) ensuing with phosphotungstic acid staining (2%, w/v) for 3 min, and CIS-PT NPs were then perceived and examined by employing them on lms of copper grid-irons. The polydispersity index (PDI), zeta potential and particle size of nanoparticles were examined using the Zetasizer ZEN3600 (Malvern, England).

In vitro proliferation assay by MTT
According to previously recorded protocols [30][31][32][33][34], a MTT assay was achieved. It is used to measure CIS-PT and CIS-PT-NPs for In vitro cytotoxicity. The cells of Caki-1 and A-498 were plated 2×10 4 cell per well in a 96-well culture plate for the attachment followed by cultured for 24 h at 37°C. 10 µL of the MTT solution was applied to each well after incubation at 37°C for 24 hours, and the 525 nm absorbance was analyzed 2 hours later by Eliza microplate reader (TECAN Sunrise, Switzerland). The percentage of the cell proliferation were calculated by using the graph pad prism software. As seen below, the cell viability (percent) was evaluated using the formula; % of cell viability = OD treated /OD control ×100 2.5. Cellular Uptake According to previously recorded protocols, a cellular uptake assay was performed. In 6-well plate at 1 ×

Flow cytometry techniques
Flow cytometry was completed to study modes of cell death over FITC-Annexin V and propidium iodide (PI) based apoptosis detection assay kit (Invitrogen, Thermo Fischer Scienti c, USA) followed by previously reported protocol [41][42][43]. The Caki-1 and A-498 cells were through the trypsinization to avoid the cell death. Trypsinized the cells and twice washed with PBS. The cells supernatant was removed and the cell pellets was suspended in a 250 µL 1 × binding buffer (from the kit) with 5 µL of FITC Annexin-V and PI was added and incubated with 37°C in dark conditions. After 10 minutes, the samples were subjected to ow cytometry analysis using FACS Canto TM II, BD Biosciences.

In vivo renal toxicity
Normal ICR mice (4-5 weeks old) were assigned to 10 classes (n = 10, 5 males and 5 females in each group) and injected intravenously at varying doses of CIS-PT-NP solution (200 µL) and CIS-PT solution thrice times every three days. Saline has been used as a control. CIS-PT treatment was given at doses of 5, 10, 20 mg/kg. CIS-PT-NPs were delivered at 5, 10, 20, 30 mg/kg doses (CIS-PT equivalents). Changes in the body weight of mice have been recorded. After having received injections of saline, CIS-PT and CIS-PT-NP, two mice in each group were randomly picked and discarded by inhalation of CO 2 . Primary organs, such as kidneys and spleens, were extracted and processed with 4 percent formaldehyde. After that, the specimens were covered in para n and sliced into 5-µm-thick strips. These slices have been stained with hematoxylin and eosin (H&E, Sigma). For the terminal deoxynucleotidyl transferase-controlled dUTP nick end labeled (TUNEL) experiment, the dewaxed and rehydrated kidney parts were incubated with proteinase K at 37°C for 15 minutes, washed with PBS once, and ushed with the TUNEL In Situ Cell Damage Detection Kit as per the manufacturer's procedure (Sigma-Aldrich). TUNEL-stained cells were counter-stained with DAB (DAKO) and imaged by optical microscopy in ten different elds for each class [44][45][46][47]. Figure 1 demonstrates the complete synthetic procedure. CIS-PT analogs conjugated with a number of successive groups at the carboxylic terminal ends demonstrate improved stability in cellular membrane due to the dehydrogenation purpose. In addition, LA hydrophilic regulator is used for the development of polymeric prodrugs. In this work, we planned and produced the CIS-PT analogs for the LA polymer chains to render the CIS-PT-LA prodrug (Fig. 1). Conjugation was achieved by coupling CIS-PT to LA under the structure of CIS-PT-LA, and the CIS-PT-LA prodrugs were ltered using thin layer chromatography (TLC) and column chromatography approaches with the high yield (80.5 %). The lipophilic design of the drug delivery systems, which create nano-assemblies, could be synthesized in water by nanoassembly approaches without the addition of any surfactants. In addition, the organic phases of the CIS-PT-LA prodrugs are absorbed in the solution of DMSO into the DD-H2O aqueous suspensions. More importantly, the elimination of excess organic solvents through double distilled water from the fabricated nanoparticles rather than prodrug accumulation. In addition, TEM observations were conducted to determine the structure of supramolecular nanoassembly CIS-PT-NPs ( Fig. 2A). The results of the supramolecular nanoassembly CIS-PT-NPs showed that the well-structured form with a sphere-shaped was ~ 80.3 ± 3.12 nm (Fig. 2B). In addition, the hydrodynamic parameter of the nanoassembly CIS-PT-NPs was 82.7 ± 2.05 nm with less poly dispersive index (PDI), as shown by the DLS method (Fig. 2C). Admirable ndings are associated with the formations of supramolecular nanoassembly CIS-PT-NPs. The stability of the CIS-PT, and CIS-PT-NPs in PBS media was observed by measuring the particle size of the CIS-PT, and CIS-PT-NPs by dynamic light scattering. Polyplexes index (PDI), precisely CIS-PT, and CIS-PT-NPs, at an nanoparticules ratio of 100:1 were organized and incubated for 30 min at 37°C in order to check broad polyplex formations (Fig. 2D-E). All the stability analysis were three times repeated.

In vitro CIS-PT release pro les
As a traditional nucleosides analog, the main shortcomings of CIS-PT as a chemotherapeutic agents contain half-life small plasma and fast cytokine deaminase deactivation. We also developed supramolecular nanoassembly CIS-PT-NPs which could help as a reservoir to keep drugs safe and avoid the release of drugs into the general blood circulation, thereby slowing down CIS-PT authorization from the human body condition. Observation of the controlled release of CIS-PT-NPs by dialyzing against PBS at RT. As seen in Fig. 2F, CIS-PT free was rapidly released from CIS-PT-NPs and plateaued at 90.2 ± 2.9 compound releases after 25 hours of incubation. In the other hand, CIS-PT-NPs demonstrated continuous drug release. Our ndings of In vitro drug leasing revealed a sluggish inhibition of the release of free CIS-PT kinetics from the CIS-PT-NPs, which is useful to enhance the half-life blood plasma of free CIS-PT and enhancing drug delivery to cancer cells.

In vitro proliferation assay by MTT
After e cient fabrication of CIS-PT-NPs, MTT experiments were conducted to determine the proliferation of CIS-PT and CIS-PT-NPs of renal carcinoma cells, with Caki-1 and A-498 cancer cells. After treating with drugs for 24 hrs, the proliferation of the Caki-1 and A-498 cells was controlled and the dose dependent curvature shows the half inhibitory concentrations (IC 50 ) (Fig. 3A-B). Positively, associated to the free CIS-PT, supramolecular nanoassembly displayed signi cantly increased cytotoxic effects in both renal cancer cell lines tested. In half-inhibitory concentrations of Caki-1 cells, the IC 50 is 11.12 ± 5.09 and 6.13 ± 1.29 for CIS-PT and CIS-PT NPs respectively. In A-498 cells, the IC 50 is 15.87 ± 2.18, and 5.16 ± 2.80 for Corilagin and Corilagin-NPs, respectively. In addition, we examined the selectivity of the CIS-PT and CIS-PT NPs shows remarkable proliferation against the CaSki and HeLa cells (Fig. 3C). The CIS-PT NPs shows remarkable proliferation against the Caki-1 and A-498 cells which may be due to the presence of the hydrophilic linoleic acid of π-conjugation chain. This may be attributed due to the hydrophobic chain of the linoleic acid which may e ciently penetrate the cell membranes. Several attempts will be made to establish the small molecule nano-assembled nanoparticles for promising anticancer agents by introducing the various lipophilic spacers on the potential small molecules.

Cellular uptake e ciency of CIS-PT NPs
We cross-linked that the CIS-PT-NPs make it possible to interact with Caki-1 renal tumor cells and ions, thereby enhancing intracellular absorption into the renal cancer cell. A confocal laser scanning microscopy (LCSM) was used to test the localizations of subcellular of CIS-PT-NPs in the Caki-1 renal cancer cell lines (Fig. 4). In the concentration 20 nM with CIS-PT-NPs with different incubation times (10, 20 and 30 min) were labelled with the DiI uorescence tracker (red color) for the interpretations, for this comparison the cells lysosome and the nucleus was labeled with the DAPI (blue color). The new yellow color uorescents were paired with DiI, and DAPI uorescence in the Caki-1 renal cancer cell line, whereas CIS-PT-NPs could be con ned with lysosome during internalization.

Morphological examination by acridine orange/ethidium bromide (AO/EB)
CIS-PT and CIS-PT-NPs were tested using a uorescent microscopic examination of acridine orange/ethidium bromide (AO/EB) stained in Caki-1 and A-498 renal cancer cell lines indicative of morphological changes (Fig. 5). Usually nanoparticles causes cell death through the apoptosis and necrosis pathways. Ironically, after 24-hour treatment with their IC 50 concentration of the CIS-PT and CIS-PT NPs, CIS-PT-NPs display a higher proportion of cell death by apoptosis pathway than free CIS-PT treatments.
3.6. Morphological examination by Hoechst-33258 staining CIS-PT and CIS-PT-NPs were tested using a uorescent microscopic examination of Hoechst-33258 staining (nuclear staining) stained in Caki-1 and A-498 renal cancer cell lines indicative of morphological changes (Fig. 6). Usually nanoparticles causes cell death through the apoptosis and necrosis pathways. Ironically, after 24-hour treatment with their IC 50 concentration of the CIS-PT and CIS-PT NPs, CIS-PT-NPs display a higher proportion of cell death by apoptosis pathway than free CIS-PT treatments.
3.6. Flow cytometry-con rmation of the apoptosis Apoptosis can be used as a signi cant impairment to the growth of a tumor cells. The primary qualitative screening methods of the AO/EB and nuclear staining methods clearly shows the supramolecular nanoassembly induce the cell death through apoptosis mode. In this quantitative experiments displays the number of cancer cell death by the ow cytometry via staining of the FITC Annexin-V and propidium iodide (PI) in the Caki-1 and A-498 renal cancer cells. CIS-PT and CIS-PT-NPs were tested using a FITC Annexin-V with the propidium iodide (PI) examination in Caki-1 and A-498 renal cancer cell lines (Fig. 7).
Ironically, after 24-hour treatment with their IC 50 concentration of the CIS-PT and CIS-PT NPs, CIS-PT-NPs with high percentage of the apoptosis than free CIS-PT.

In vivo renal toxicity
In vivo systemic toxicity induced by CIS-PT has hindered its therapeutic use [48]. To con rm if this CIS-PT-NPs scaffold transforms high toxic CIS-PT to stable nanotherapy, we intravenously delivered stock solution CIS-PT-NPs to normal ICR mice. After injection of CIS-PT-NPs or clinically used CIS-PT solution, we measured the shift in body weight of the mice. Normal ICR mice (4-5 weeks old) were assigned to 10 classes (n = 10, 5 males and 5 females in each group) and injected intravenously at varying doses of CIS-PT-NP solution (200 µL) and CIS-PT solution thrice times every three days. Saline has been used as a control. CIS-PT treatment was given at doses of 5, 10, 20 mg/kg. CIS-PT-NPs were delivered at 5, 10, 20, 30 mg/kg doses (CIS-PT equivalents). The healthy ICR mice (n = 10, 5 males and 5 females in each group) and injected intravenously injected with 5, 10, 20, or 30 mg/kg (CIS-PT-equivalents) thrice times every three days. Even 5 mg/kg of CIS-PT was well received by mice (~ 4.7% body weight reduction) as larger concentrations (10 and 20 mg/kg) resulted in substantial weight loss (Fig. 8). However, CIS-PT-NPs signi cantly increased the therapeutic e cacy of the parental CIS-PT. After these three injections of elevated doses of drugs (e.g. 30 mg/kg of CIS-PT-equivalents), no obvious effects on body weight were found, suggesting little or no drug toxicity.
Renal toxicity is a signi cant adverse side effect of CIS-PT, which has reduced dosage enhancement in clinical practice. We have also checked if this CIS-PT-NPs scaffold is less dangerous to the tissues. Healthy ICR mice were treated with double injections of CIS-PT-NPs (5, 10, 20 and 30 mg/kg with CIS-PTequivalents), and CIS-PT (5, 10 and 20 mg/kg) was used as a standard. The animals were sacri ced on day 6 of post-injection and the organs were exposed to histological immunohistochemistry (H&E) and TUNEL staining. Lower damage to kidneys and spleen caused by the use of CIS-PT-NPs has also been reported by H&E staining (Fig. 8). As seen in Fig. 9, kidney segments in CIS-PT-treated mice displayed substantial cell death activation, while only marginal nephrotoxicity was identi ed in mice given CIS-PT-NP at larger concentrations up to 30 mg/kg. Together to, such ndings show that CIS-PT-NPs protect the kidney from CIS-PT-induced dysfunction in animals, which may potentially favour humans when evaluating medical properties.

Conclusion
We have effectively engineered and illustrated the lipophilic and fast metabolic CIS-PT prodrugs for a more pharmacologically effective nano prodrug. We nd out that supramolecular nanoassembly of CIS-PT nanoparticles display improved controlled drug release and enhanced extracellular environment uptake to possibly resolve cancer cell aggregations by EPR. After a successful development, we tested the MTT of CIS-PT and CIS-PT-NP nanoparticles against Caki-1 and A-498 renal tumor cell lines. In addition, the morphological changes experiments such as acridine orange/ethidium bromide (AO-EB), Hoechst-33258 staining results shows that the supramolecular nanoassembly of CIS-PT nanoparticles induce apoptosis in renal cancer cells. Further, we have con rmed the apoptosis by owcytometry techniques. More admirably, CIS-PT-NPs demonstrated decreased toxic effects relative to CIS-PT. As such, the new CIS-PT prodrug-assembled scaffold can be used in patients with compromised renal function. Ultimately, this work offers a simple approach to producing cost-effective and healthy platinumbased nanotherapy and deserves further application to clinical practice.       Flow cytometry examination were performed to con rm the apoptosis of Caki-1 and A-498 renal cancer cells. The Caki-1 and A-498 cells were treated with CIS-PT and CIS-PT-NPs with IC50 concentration for 24 hrs and stained by FITC-Annexin V and PI for ow cytometry examination.