Formulation of Folate-conjugated, Doxorubicin-loaded Human Serum Albumin Nanoparticles for Promotion of Gene Expression Associated with Apoptosis in Renal Cell Carcinoma

In this study, we developed a new, ecient targeting and pH-responsive formulation using folate (FA)-conjugated human serum albumin (HSA) nanoparticles to deliver doxorubicin (DOX) drug to renal cell carcinoma. FA was conjugated to HSA, and DOX was loaded into obtained FA-HSA NP using the desolvation method. FA-HSA-DOX NPs were examined using zeta potential and (DLS) measurements, dynamic light scattering, scanning electron microscopy, atomic force microscopy, FTIR, and UV-Vis Spectroscopy. Drug liberation rate, toxicity/viability rate, and the apoptosis/necrosis effect of different DOX, FA-HSA, and FA-DOX-HSA treatments were evaluated using release prole study, MTT assay, and ow cytometry methods, respectively. The expression of BAK, BCL2, BAX, and BCL-XL genes in cancer cells after treatment was analyzed via real-time polymerase chain reaction (PCR). The average size, zeta potential, and polydispersity of FA-DOX-HSA NPs were 200 ± 9 (nm), − 47 ± 7 (mv), and 0.012, respectively. This formulation showed pH-dependent drug release, high drug loading performance, sustained ability of drug release. The notable toxic properties and high apoptosis-stimulation ability of the FA-HAS-DOX NPs in RCC-GH cell lines demonstrated long-term anticancer ecacy. Also, real-time PCR results showed an increase in BAX expression and a decrease in BCL-2 L1 and BCL-XL signicantly. Our ndings indicate that FA-HSA NPs have no cytotoxic effect on cancer cells (RCC-GH) and healthy cells (RPTEC/TERT1). Our study provides a simple and ecient FA-HSA carrier with intrinsic biocompatibility for DOX drug delivery


Introduction
DOX is an e cient anticancer drug that belongs to the anthracycline antibiotics and is commonly used alone or in combination with other chemotherapy agents in the treatment of a broad range of cancers such as lymphoma, leukemia, and also breast, bladder, and kidney cancers [1][2][3][4][5][6]. However, the inability to select target cells, the harmful adverse effects such as cardiotoxicity, and the emergence of drug resistance remain signi cant limitations associated with DOX administration [7,8]. Drug delivery-based nanoparticles (NPs) (especially natural polymeric NPs (have gained great attention because of their nanoscale properties and their enhanced permeability and retention of anticancer therapeutics [9][10][11][12].
High biocompatibility of natural materials such as protein-based NPs can be used as ideal drug carriers because of their distinctive characterizations such as amphiphilic, shelf life, the possibility of surface modi cation, and biodegradability [13]. HSA is the largest constituent of plasma proteins that contributed to the upkeep of osmotic pressure. It acts as a biocompatible, non-toxic, and biodegradable natural polymeric material. This molecule comprised amino acids connected to peptide bonds that make it possible for embedding and drug loading. HSA enhances drug stability and permits it to release into cancer cells through cellular uptake, which makes it an ideal platform to fabricate NPs for drug delivery systems [14,15]. The folate receptor is known as a tumor cell biomarker because it can overexpress on the surfaces of various cancer cells, encompass renal, lung, colon, brain, uterine, cervix, and breast [16].
However, it is rare to nd this protein on the surface of healthy cells [17]; this difference can be used to target malignant cells to improve drug delivery. In this work, to improve targeted treatment to deliver DOX drug in human clear epithelial carcinoma (RCC-GH) cells in vitro model, we have focused on fabricated folic acid-coated human serum albumin NPs (HSA-FA). The prepared FA-HSA-DOX NPs were evaluated for DOX drug release, and their anticancer activity on both Human Kidney clear cell carcinoma (RCC-GH) and human renal proximal tubule (RPTEC/TERT1) cells were examined. We have also investigated the cellular proliferative changes and mRNA gene expression of pro-apoptotic gene BAX and anti-apoptotic gene BCL2 after the treatment of RCC-GH cancer cells with FA-HSA NPs that comprise DOX.

Preparation of folate-DOX-loaded HAS (FA-DOX-HAS) NPs
FA-functionalized HSA NPs were prepared at ambient conditions using an ethanol desolvation method [18]. Brie y, 50 mg of HSA was added to 5 mL of 10 mM NaCl solution under 800 rpm stirring at room temperature. The solution was stirred continuously for 15 min and then its pH was titrated to 8.5 using 1 N NaOH for 5 min stirring. The ethanol was added dropwise to a desolvating factor of HSA solution under continuous stirring until the HSA solution became turbid (∼1-2 mL). To form stable HSA NPs 10% glutaraldehyde (GA) as a cross-linking agent was added to the obtained turbid HSA solution. The centrifugation of this solution was performed at 14000 rpm for 15 min. Afterward, the NPs sedimentation was rinsed with distilled deionized water (DDW). Then, the rinsed NPs were suspended in an equal volume of PBS. FA dissolved in DDW (followed by NaOH addition) was added to the resulted NPs solution. The mixed solution was gently vortexed for 1 min to allow the electrostatic binding of FA on the surface of HSA NPs. For the nanoformulation of drug-loaded FA-HSA NPs, DOX was added to 1 mL FA-HSA solution and kept in constant stirring for 5 h, followed by dropwise ethanol addition.

Characterization
A DLS (Nanopartica SZ-100; HORIBA Ltd, Kyoto, Japan) was used to measure the polydispersity, size, and surface charge of NPs. Particle size distribution, zeta potential, and polydispersity index of NPs were measured using deionized water (1 mg/1 mL) through a Zeta sizer (Malvern Instruments Ltd., Malvern, UK). The morphological features were examined using SEM (Philips XL30) and AFM (Nanoscopy Digital) Instruments.

Fourier-transform infrared spectroscopy (FTIR)
FTIR is an effective technique for evaluating the structure and formation of NPs. A spectrometer (Nicolet, Madison, WI, USA) was applied to record the FTIR spectra from vacuum-dried samples. The FTIR resolution was set at 4 cm −1 and used to t the 400-4000/cm spectrum scope.

Ultraviolet-visible (UV-vis) spectrophotometry
The appropriate combination of drugs and the successful combination of target agents plays a key role in building nanocomposites. UV-vis experiments were performed to con rm the conjugation of FA, DOX, and HSA molecules on FA-DOX-HSA NPs. The absorption spectrum of FA-DOX-HAS NPs was measured using a UV-vis spectrophotometer (Shimadzu, Tokyo, Japan) with a 1 cm quartz cuvette to detect absorption range at different intervals between 200 and 700 nm.

Encapsulation e ciency
The NP solution was centrifuged for 20 min at 14000 rpm. Drug-loaded NPs (FA-DOX-HSA) were estimated, and the free unloaded DOX drug in the supernatant was measured using a UV spectrophotometer. The following equation was applied to measure encapsulation e ciency: Encapsulation e ciency (%) = [(Total drug − Free unloaded drug)/Total drug] Eq. (1)

In vitro determination of DOX drug release
To measure the DOX drug release from the prepared nanosystem FA-DOX-HSA, under in vitro conditions, membrane-drug diffusion studies were utilized. Brie y, 1 mL of FA-DOX-HSA suspension was transferred in a dialysis bag and then immersed in 100 mL phosphate buffer (pH = 7.4) and citrate buffer (pH = 5.4) and incubated at 37°C within bath shaking. One milliliter of FA-DOX-HSA suspension was drawn out, freeze-dried, and dissolved in 2 mL of methanol. Fluorescence spectroscopy (Cary 100 BIO UV-vis spectrophotometer, Varian, CA, USA) at 450 nm was used to determine the DOX amount release from FA-DOX-HSA formulation.
The following equation was used to calculate the DOX drug release: where R is the nal drug release (%), V is the volume of each sample, V 0 is the initial volume of drug, Ci and Cn are the DOX concentrations, i and n are the sampling times, and m drug is the mass of DOX in NPs.

Cell culture
RCC-GH and RPTEC/TERT1 cell lines were purchased from ATCC (UK). All cells were plated and cultured in DMEM enriched with 10% FBS and glutamine (2 mM) and incubated in 37°C and 5% CO 2 atmosphere.

Cellular internalization
The uorescence emission spectrum of DOX was inspected in various biological medium to evaluate the ability and activity of the FA-DOX-HSA formulation in DOX protection. For this purpose, the cells were treated with 25 μg mL −1 FA-DOX-HSA. Additionally, FA-HSA and DOX with the same concentration served as references. The imaging process was performed using a uorescence microscope (Nikon Eclipse TE2000-U) in the range of 420 to 70 nm.

MTT assay
RCC-GH and RPTEC/TERT1 cells (in cultured media) were seeded into 96-well plates at a density of 1 × 10 5 cells per well and incubated overnight to make the cells adhere to the wells. Then, the media were changed with achieved concentrations of FA-HSA, DOX, and FA-DOX-HSA NPs that were dissolved in a medium with a range of 10 to 60 μM. Following 24 and 48 h of treatment. The toxicity was examined after adding a 5 mg/mL MTT solution (a further 4 h incubation). Then, the media were removed and 100 μL of DMSO was added to each well. The absorbance was measured at 570 nm, on 690 nm wavelength using a microplate reader (Tecan Group Ltd., Männedorf, Switzerland).

Flow cytometry
Cell apoptosis was conducted using ow cytometry analysis based on Annexin V-FITC Apoptosis Detection Kit (Biovision, Inc.) 1.0 × 10 5 cells per well were seeded into six-well plates, and after 1 day of plating, the cells were treated with FA-DOX-HSA, void DOX, and FA-HSA NPs and incubated for 48 h. PBS was used to wash the cells twice and then trypsinized, and then, the cells were suspended in Annexin binding buffer using Annexin V-FITC and propidium iodide stain.

Apoptosis assay by quantitative real-time PCR
After treating the RCC-GH cells with DOX, FA-HSA, and DOX-loaded FA-HSA NPs, they were transferred to a six-well plate at a density of 1 × 10 5 cells/mL and cultured in a humidi ed atmosphere for 48 h. RNA was extracted from cells after 48 h of treatment by using TRIzol according to the manufacturers' protocol (Invitrogen Life Technologies, UK). Complementary DNA (cDNA) synthesis was achieved based on the protocol of the RevertAid M First-Strand cDNA Synthesis Kit. The reaction was conducted in iCycler thermal cycler (Bio-Rad, Hercules, CA, USA) following the protocol cycle that involved 25℃ for 6 min, incubation at 42℃ once more for 1 h, and then heating at 75℃ for 5 min. cDNA is directly used in the real-time PCR. Table 1 mentions the primers for targets and endogenous genes, designed using software primer express. Ampli cation reactions contained 5 μL of cDNA, 10 μL of the SYBR Green-I dye (Applied Biosystems, USA), and 0.5 μL of each of the speci c primers. Primers' concentration in the nal volume of 20 μL was 100 nM. Real-time PCR was performed as follows: 50 cycles initiated at 95°C for 10 min and then at 95°C for 15 s and 60°C for 1 min; the real-time PCR success was evaluated with the melting curve analysis.

Statistical analysis
Statistical analysis was conducted using SPSS software (version 22), and data were expressed as unpaired Student's t-test, standard error of the mean (mean ± SEM), and analysis of variance (ANOVA) with *p < 0.05 considered as statistically signi cant.

Characterization of FA-HAS-DOX NPs
Recently, various researches have focused on HSA as a promising agent for carrying anticancer drugs. Although the utilization of organic solvents to prepare NPs is preferred, many materials are di cult to dissolve in both water and organic solvent. One way to prepare such NPs without applying organic solvents is to use ethanol [23].
In the present study, the ethanol desolation method was used to produce FA-HAS-DOX NPs, in which ethanol was used as a desolvating factor and GA as a cross-linking media. GA is a water-soluble, lowcost, and bifunctional substance with notable reactivity. The cross-linking process among protein structures happened by the two carbonyl groups of GA and due to the nucleophilic attack of the ε-amino groups in lysine and arginine residues. The NPs size will not change due to GA reaction process but the surface charge is signi cantly affected. [24,25]. Thus, GA was used as the cross-linking, stabilizing agent in aqueous and cellular media.
The dispersion of HSA in water was performed, and ethanol was used as the dehydrating agent after pH was adjusted to alkaline value. This method is regarded as a simple and low-cost technique and produces a lower aggregation rate with uniform NP distribution than those in other methods [26]. According to a study conducted by Lomis et al., the particle size might be moderately affected with an alteration of pH in the range of approximately 100-200 nm [27]. DLS and Zeta data of synthesized FA-DOX-HSA exhibited that the sample provided an overall diameter size of 200 ± 9 nm, which is consistent with previous studies [28]. The polydispersity was 0.012, which presents a negligible diversity of the NP size and a − 47.2 mV charge ( Fig. 1A and B), which shows high stability and is desirable for different biomedical applications [29]. Besides, Li Q et al. indicated that the particle size of hydroxy-camptothecin encapsulated by HSA and modi ed by FA was 233.9 ± 1.2 nm and the zeta charge was − 25.23 ± 2.98 mV [25]. The results provided by SEM together with AFM agreed with the results obtained with Zeta analysis, indicated that FA-DOX-BSA NPs have discrete spherical shapes that can be detached from their neighbor with homogeneous surface density ( Fig. 2A and B). The loading activity of FA-DOX-HAS NPs was 79%.  Figure 3 (i) shows an infrared spectrum peak of bare FA (the stretching vibration of the benzene ring skeleton at 1500/cm − 1 ). The second peak is located at 1602 cm − 1 , which corresponded to the spectrum of the bare DOX, as shown in Fig. 3 (ii). In Fig. 3 (iii), the HSA absorption peak was 1790 cm − 1 , due to a large number of amino acids compound in this molecule. The infrared spectrum of FA-HSA NPs as shown in Fig. 3 ( ) contains the characteristic peaks of FA and has a peak at 3410 cm − 1 . The infrared spectrum of the FA-DOX-HSA nanoformulation has an absorption peak at 1651 cm − 1 and 3324 cm − 1 . A small peak appeared in the infrared spectrum of FA-HSA-DOX NPs (Fig. 3(v)), indicating the presence of a certain amount of DOX on the surface of FA-HSA NPs.

UV-vis spectroscopy
UV-vis spectroscopy is used to further test the encapsulation of FA, HSA, and DOX in the NPs. Results indicated the spectral data details of the energy band gaps and optical transition to prove the anticancer drug bonding to the FA-HSA. The UV-vis spectra of pure FA, HSA, and Dox and their NP counterparts were signi cantly different. The spectra exhibited an absorbance peak at 550, 340, and 310 nm, respectively, whereas both a mixture of free drugs and FA-DOX-HSA NPs exhibited a broad absorbance peak at 510 nm (Fig. 4).  3.6. Flow Cytometry Figure 8 shows the cell apoptosis analysis. The results exhibited that the treatment of RCC-GH cells with bare nano-carrier or void DOX drug did not present noticeable apoptosis induction after 24 h of treatment. Conversely, the level of RCC-GH cells affected by apoptosis was increased remarkably when subjected to FA-DOX-HSA nanoformulation. These ndings con rm that FA-DOX-HSA has notable potential to kill RCC-GH cells using DOX and noticeably induce programmed cell death in cancer cells. Moreover, it was not possible to make a distinction between these two patterns of cell death only by using the progressive loss in the integrity of the plasma membrane as an indicator [32]. The use of NPs as capsules for anticancer drugs can facilitate their intake by the cells and the lysosomes, leading to more highly induced cytotoxic activity [33][34][35][36]. One previous investigation indicated that increasing the development of intracellular ROS could be responsible, at least partially, for the killing effect of anticancer drugs [32]. The incubation of cancer cells with NPs has been associated with altered cell proliferation and induced apoptosis, in uenced by changes in ROS development levels [19]. However, events such as increased morphological apoptotic alterations and DNA damage may be due to elevated ROS concentrations and associated mitochondrial membrane alterations.

Real-time PCR and gene expression pro le
Bcl-2 family proteins can activate Bax, Bak (pro-apoptotic) and Bcl-2, Bcl-xl (anti-apoptotic) cells inhibition. Researchers have indicated that the ratio changes to pro-apoptotic and anti-apoptotic proteins regulated cellular apoptosis [20,21,37]. N Pilco-Ferreto reported the requirements of BCL2 expression in response to DOX [38]. We have investigated the expression of the mentioned genes in RCC-GH after treatment with FA-DOX-HSA NPs in comparison with the FA-HSA and void DOX, in which after treatment, the FA-DOX-HSA signi cantly decreased the expression of Bcl-2 and Bcl-xl of the control group (untreated). Moreover, FA-DOX-HSA considerably upregulated the expression of BAK and Bax genes of healthy levels (P < 0.01) (Fig. 9).

Conclusions
The results of the study concluded that the FA-DOX-HSA nanoparticles were successfully developed and demonstrated the effective cellular uptake of DOX anti-cancer drug, on the other hands The conjugates between FA-HSA nanoparticles and DOX drug are of good high activity with pH-dependent drug release, these conjugates act as synergistic biocompatible treatment, with low cellular toxicity effect when compared to DOX drug alone, that could be used for future cancer therapy.

Declarations
Con icts of Interest: The authors declare no con ict of interest. Tables   Table 1: The primer sequences that were utilized to amplify the genes.

Gene
Forward primer sequence Reverse primer sequence Ref.