A convergent fabrication of programmed pH/reduction-responsive nanoparticles for efficient dual anticancer drugs delivery for ovarian cancer treatment

Abstract A nanoparticle-based drug delivery technology could develop combination cancer therapy more effectively. However, because of inadequate drug delivery into tumor cells, the cancer therapeutic efficiency of nanomedicines is diminished. PEGylated poly(α-lipoic acid) copolymers with (mPEG-PLA) were fabricated and used as pH/reductive responsive nanovesicles to administer Gefitinib (GFT) and doxorubicin (DOX) for the treatment of ovarian cancer. The amphiphilic polymers mPEG-PLA may be efficiently incorporated on DOX and GFT to fabricate DOX and GFT coloaded nanoparticles (DOX@GFT-NPs) and self-assembled into an aqueous solution. The DOX@GFT-NPs released more DOX and GFT after being prepared to respond to pH and reduction stimuli. The outcomes of confocal laser scanning microscopy and flow cytometry findings, the SKOV3 ovarian cancer cells quickly fascinated the dual drugs-coloaded nanoparticles and drugs released intracellularly accumulated. DOX@GFT-NPs triggered cell death and demonstrated synergistic therapeutic benefits in SKOV3 cells. Results showed that the nanoparticles efficiently trigger apoptosis in SKOV3 ovarian cancer cells using morphological staining (acridine orange/ethidium bromide (AO/EB) and HOECHST 33342 nuclear staining). These outcomes show that using pH and reduction stimuli on mPEG-PLA copolymer to treat ovarian cancer is a promising approach.


Introduction
Developing a technology capable of high-specificity targeted administration of anti-neoplastic medications would be a huge breakthrough in cancer in general and ovarian cancer [1][2][3].Although the circulatory system can distribute medicine to every cell in the body, getting the drug selectively within the tumor cell across its membrane without damaging the healthy cells remains challenging.Intraperitoneal (IP) administration by a surgically implanted catheter has demonstrated better survival rates in ovarian cancer [4].However, catheter problems and toxicity have hindered the general use of this invasive delivery form [5]. Current research aims to work around these limiting limitations by employing nanoscale systems.As immunological reagents, monoclonal antibodies are often utilized to detect the tumor-specific biomarker, while the nanoscale control further increases the specificity and targeted drug delivery capabilities in general [6].Nonetheless, despite the significant advances in this sector throughout the previous decades, the capacity of targeted delivery with appropriately high specificity (to tumor cells) remains an essential hurdle to finding a cancer treatment [7,8].
Oral gastroprotective systems have been developed due to conventional systems' failure to maintain gastric retention [9].Such delivery methods were created to be kept in the upper gastrointestinal system for an extended period while slowly and consistently releasing the medication [10].Drug bioavailability can be increased due to the prolonged interaction of gastroprotective systems with the absorbing membrane [11].These methods are increased therapeutic efficacy, decreased drug loss, increased drug solubility in situations where drug solubility was low in a high pH microenvironment, and advantages from administering drugs that act regionally in the stomach and duodenum [11][12][13].
Due to their small size and the advantageous and adjustable features of the polymeric nanoparticles.Submicron (100-1000 nm) nanoparticles are often generated from polymers, lipids, viruses, and inorganic materials [14][15][16].Several polymers have been employed for cancer therapy, including PLGA, PLA, and others approved by regulatory bodies.Abraxane is one of the nanotherapeutics used to treat metastatic ovarian cancer [17].Previous investigations have revealed that therapeutic drugs are removed and destroyed, creating undesired biological behavior and toxicities [18].Consequently, nanoparticles have several drawbacks, including low bioavailability, instability, toxicity, and limited tissue dispersion.Only 0.7% of the given medication formulations reached solid tumours in a 10-year nanoparticle-based drug delivery systems study [19].As a result, it is necessary to design nanoformulations with fewer adverse effects [20].Biotherapeutic drugs may be administered solely in their cytoplasm to cancer cells using nanoparticle-based therapy.Several investigations have shown that appropriate carriers may effectively transport drugs to the target location despite physical obstacles.One of the most efficient adaptations combines polyethylene glycol (PEG) and polylactic acid (PLGA).PEG, like PLGA, has been authorized for diagnostic devices by the FDA and the European Medicines Agency [21][22][23].To attain maximum therapeutic effectiveness, nano-delivery systems and drug release modification need additional nanoparticle crosslinking for specific drug targeting [24].
An anticancer drug called Gefitinib (GFT) is the first specific inhibitor of the tyrosine kinase region of the epidermal growth factor receptor (EGFR) tyrosine-kinase domain [25].The transmembrane glycoprotein epidermal growth factor has unique tyrosine kinase receptors that govern high cellular proliferation and expression levels in cancer [26].Solid tumors, including ovarian, lung, colorectal, and brain, will likely exhibit more elevated EGFR.To suppress cancer cell proliferation, GFT inhibits the kinase activity of wild-type and specific activating mutations of the EGF receptor, thereby blocking tyrosine residues linked with the receptor's autophosphorylation, preventing further downstream signaling [27][28][29].
In cancer treatment, doxorubicin (DOX) is a first-line drug that targets DNA in nuclei.When treating ovarian cancer, DOX has a low nuclear delivery efficiency.DOX treatment for ovarian cancer may benefit from facilitated delivery [30].Liposomes are a commonly used drug delivery technology because of their high loading capacity, superior biocompatibility, and biodegradability.Drug carriers for DOX that have been changed, such as galactosylated liposomal lipases or lyso-thermosensitive liposomes, have been shown to boost therapeutic efficacy.Some preclinical studies had shown enhanced treatment effectiveness for ovarian cancer when DOX was delivered via superparamagnetic iron oxide (SPIO) nanoparticles and hematoporphyrin (HP)-modified DOX-loaded nanoparticles (HP-NPs) [31].Some drawbacks remain, such as the intrusiveness, off-target impact, limited biocompatibility of this method, and low efficiency in the long term [32].There is still a need to design a DOX delivery method that is more precise and effective in treating ovarian cancer with precision.A significant step forward in cancer treatment will be made possible by developing novel, nuclear-targeted drug delivery systems (DDSs), which can destroy cancer cells more quickly and effectively than existing treatments [33][34][35].
In recent times, nanoparticle-based drug delivery systems have been extensively used to enhance the selectivity, reduce the side effects, and enhance the anticancer effects of chemotherapeutic drugs by enhanced permeability and retention (EPR) effect.Several reports were published using Gefitinib (GFT) and doxorubicin (DOX) with different nanocarriers [36][37][38].But some of them show several adverse effects on various cancer cells, and this is the first paper to examine ovarian cancer cells.To overcome these issues, we established PEGylated poly(α-lipoic acid) copolymers with (mPEG-PLA) which were fabricated and used as dual-responsive (pH/reduction) mediated nanovesicles to administer Gefitinib (GFT) and doxorubicin (DOX) for the treatment of ovarian cancer (Figure 1).The nanoparticles may be coated with the GFT and the DOX through electrostatic and hydrophobic interactions, ensuing in coloaded polymeric nanoparticles (DOX@GFT-NPs).Following cellular internalization in an intracellular acidic environment, the protons shifting of mPEG-PLA carboxyl groups and DOX amino groups inhibited electrostatic and hydrophobic relations among the mPEG-PLA and the coloaded potential antitumor dual drugs.Furthermore, nanoparticle detachment was initiated by the breakdown of the PLA strength.Both processes resulted in a decrease in pH and the release of GFT and DOX in SKOV3 cells (Figure 1).5-diphenyl tetrazolium bromide (MTT) was obtained from the Sinopharm Chemical Reagent Co., Ltd.(Shanghai, China).Doxorubicin hydrochloride (DOX.HCl) and Gefitinib (GFT) were purchased from were obtained from Chemsoon Co., Ltd.(Shanghai, China).Fetal bovine serum (FBS, Gibco) and Dulbecco's modified Eagle's medium (DMEM, Gibco), and HOECHST 33342 were purchased from ThermoFisher Scientific (Shanghai, China).All the chemicals mentioned above and reagents were used as received.Human ovarian cancer (SKOV3) was purchased from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences (CAS, Shanghai, China).SKOV3 cells were grown as a monolayer in a humidified incubator in Dulbecco's modified eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) at 37 °C in a 95% air, 5% CO 2 .

Characterisation of nanoparticles
The nanoparticles' transmission electron microscopy (TEM) images were recorded using a transmission electron microscope (Tecnai F20, FEI company).Dynamic light scattering (DLS) and ζ-potential results were recorded on a Zetasizer Nano ZS instrument (Malvern).Fluorescence microscopy images were obtained on an Olympus CK53.The high-performance liquid chromatography (HPLC) was performed on a Shimadzu LC-2010A HT liquid chromatography.

Preparation of PLA and mPEG-PLA
Self-polymerization of α-Lipoic acid (α-LA) occurred when the reaction temperature rose over its melting point without an initiator.The α-lipoic acid (2.02%, 5 mmol) was added to a dry flask and stirred at 85 °C for 2 hrs with nitrogen flow.The crude product was dissolved in 25 mL of THF and then poured into 200 mL of ether to form a white, sticky solid.Vacuum drying and filtering fabricated the final product (1.0 g, 49.3%).
To fabricate the mPEG-PLA, mPEG was conjugated to the PLA backbone and grafted onto the PLA backbone.In the first step, 831 mg of PLA (one mole) was dispersed in 25 mL of THF, followed by a combination of mPEG and EDC.HCl and DMAP were mixed in 25 mL of DMSO.It was then dialyzed against deionized water for three days after 48 hrs of agitation at room temperature, using a dialysis bag (MWCO = 3500 Da).After lyophilization, the final product, mPEG-PLA, was a white powder (2.4 g, 78.3%).

Construction of mPEG-PLA and DOX@GFT-NPs
In the synthesis of the nanoparticles, mPEG-PLA (200 mg) was dissolved in DMSO (10 mL) and then slowly mixed with 75 mL of double-distilled water, constantly stirring (NPs).After 80 min, the DMSO solution was dialyzed in contrast to DI water for 12 hrs.After lyophilization, the nanoparticles were obtained.
A technique for constructing drug-loaded nanoparticles was previously published [39].DMF (7 mL) was dispersed in 100 mg of mPEG-PLA and 30 mg of DOX.HCl.DOX@ NPs were fabricated using HCl, whereas GFT@NPs were used with GFT (30 mg), progressively added to 100 mL of quickly swirled aqueous solution in the darkness to get the required products.After being dialyzed for 90 min in a dialysis bag, dialyzed for 15 hrs against DD-water.GFT@NPs, DOX@NPs, and DOX@GFT-NPs were developed after lyophilization.Fluorescence spectrophotometry was utilized to calculate drug loading efficiency (DLE) and drug loading content (DLC) in DOX and GFT, which were intended as follows: Loaded drugs weights Drugs Loadednanoparticles weights % / u100 f for DLC.
Loaded drugs weights Feeding drug weights %for DLE / .u100

Drug release profile
For drug release testing, a dialysis membrane bag (MWCO = 4000 Da) was packed with 2 mL of DOX@GFT-NPs and incubated in the dark at 37 °C with continuous shaking.GSH was used as the drug release medium, and DOX was dispersed into PBS at pH 5.5 and 7.4.Tween-80 (1%) was added to the GFT release medium with a pH of 7.4.At a pH of 5.5, PBS with Tween-80 (1%) was also used.Tween 80 (1%) with pH 7.4 and GSH were used in PBS with Tween-80 and GSH (5 mM).Fluorescence estimate (480 nm excitation and 592 nm emissions) and HPLC were calculated to investigate the discharge of GFT and DOX [40].

SKOV3 cellular internalizations of DOX@GFT-NPs
To investigate the cellular internalization of DOX@GFT-NPs, SKOV3 cells were seeded in a 24-well plate (1 × 10 4 cells per well) and cultured overnight.DOX@GFT-NPs were incubated with cells at a final DOX concentration of 0.1 μg/mL.After 3 or 6 hrs of incubation, cells were stained with DAPI and observed under a confocal fluorescence microscope (CLSM, Olympus FV1000).
To investigate the macrophage-escaping ability of DOX@GFT-NPs, SKOV3 cells were seeded in a 24-well plate (1 × 10 4 cells per well) and cultured overnight.Cells were incubated with DOX-free, DOX@NPs, and DOX@GFT-NPs at a final DOX concentration of 0.1 μg/mL.After 6 h of incubation, cells were washed, trypsinized, and resuspended to determine the fluorescence intensity of DBNP through flow cytometry (FACS, Becton-Dickinson Biosciences, Drive Franklin Lakes, U.S.).

In vitro cytotoxicity
In vitro cytotoxicity of newly fabricated nanoparticles and free drugs were assessed by MTT (3-(4, 5-cimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) assays against the SKOV3 cells.Briefly, the cells were seeded in a 96-well plate in which cell density was 1.0 × 10 4 cells per test well and cultured overnight at 37 °C in a 5% CO 2 incubator.The samples were added to the respective test well, and the cells were cultured in 5% CO 2 .The concentrations of the samples were 0.625, 1.25, 2.5, 5, and 10 μg / mL, respectively.After incubation for 48 or 72 hrs, the MTT assay was performed to quantitatively measure the relative cell viability [41][42][43][44].The absorbance was measured at 472 nm using a microplate reader (BioPak plate reader).Cell viability of the SKOV3 cells was calculated as follows: (A sample /A control )×100%.

Morphological assessment
The AO/EB staining process checked for morphological evidence of SKOV3 apoptosis in the cells treated with the samples at IC 50 concentrations.AO and EB were added to PBS in a 1:1 ratio and incubated for 5 min before the unbinding dye was removed by washing the cells with PBS.Cells treated for 24 hrs with samples were labeled [45][46][47][48].
Fluorescence microscopes (Olympus CK53) and the percentages of cell apoptosis were assessed.
Different time intervals, like 24 hrs, were used to treat SKOV3 ovarian cancer cells in 6 well plates with samples with IC 50 concentrations.The cells were extracted by trypsinization and washed twice with PBS at each time point.The cells were then fixed for 20 min in 4% paraformaldehyde and re-washed before being stained for 20 min at 37 °C with HOECHST 33342 (10 µg/mL).After rinsing the plates with methanol and PBS, the plates were focused on using fluorescent microscopy (Olympus CK53) to determine any nuclear structural alterations and apoptotic bodies generated by substances under blue channel fluorescence [49][50][51].

Statistical test
All the experiments were repeated at least thrice.The experiment results were expressed as means ± standard deviation (SD).Statistical significance was evaluated using a student t-test when two groups were compared.Data were analyzed by GraphPad Prism 8 software.*p < 0.05 was statistical significance.

Construction of DOX@GFT-NPs
To construct DOX@GFT-NPs, a PEGylated-PLA copolymer termed mPEG-PLA.Nanoparticles coated with mPEG have a longer circulation time because they limit inappropriate protein binding and physiological recognition.Intracellular GSH may produce a reduction-responsive drug release profile by disrupting the disulfide-bond-containing PLA backbone.In addition, hydrophobic and electrostatic interaction between carboxyl groups on the hydrophobic PLA framework may be used to co-load GFT and DOX drugs, which would be released when the intracellular pH becomes acidic.The fabrication of mPEG-PLA is depicted in Figure 1.Ring-opening polymerizing LA first made PLA at 98 °C for 2 hrs.
Transmission electron microscopy (TEM) (Figure 2A) reveals nanoparticles' consistent dispersal of spherical fragments.For hydrophobic and electrostatic interactions, synthetic amphiphilic mPEG-PLA was used to fabricate the DOX@GFT-NPs.It was found that the nanoparticles size of DOX@GFT-NPs was about ~90% larger than that of mPEG-PLA (NPs), and DOX-free and GFT-free coloaded nanoparticles were found to be ~90 nm (Figure 2B).The polydispersity index and zeta potential of the DOX@GFT-NPs show 0.135 and −12.25 mV, respectively.In DOX@GFT-NPs, drug loading efficiency (DLE) was 23.2% and 81.68%, respectively, whereas drug loading content (DLC) was 5.1% and 8.8%.In the abovementioned examination, mPEG-PLA was loaded with dual anticancer drugs DOX and GFT concurrently with excellent efficacy.The DLS measurements examined the colloidal stability of the mPEG-PLA, GFT@NPs, DOX@NPs, and DOX@ GFT-NPs with an aqueous solution and DMEM cell culture medium at 24 hrs with different incubation periods at room temperature.The outcomes of particle size, polydispersity index, and zeta potential indicate strong interactions for the mono-dispersion and long-term stability of the polymeric core colloidal suspensions with anticancer drugs (Figure 3A-C).

Drug release profile of DOX@GFT-NPs
Due to the abnormal intracellular environment of the tumor compared to normal tissues, such as acidic pH levels of endosome and lysosome (5.0-5.5),high-level glutathione (GSH) concentration (2-10 mM), and oxidative microenvironment in mitochondria due to high concentration of H2O2, a series of hybrid materials prepared for controlled drug release at tumor location were always pH or GSH stimulus-responsive.Developing a smart hybrid material that both pH and GSH can control concurrently is still important for enhancing the curative efficacy and minimizing the adverse effects of cancer chemotherapy.To examine the reduction/pH dependence of drug release, the time-responsive release of DOX and GFT from DOX@GFT-NPs was achieved in PBS at pH 7.4, PBS at pH 5.5, and PBS at pH 7.4 comprising 5 mM GSH to mimic the normal extracellular environment, acidic endolysosomal circumstance, and the reductive cytosolic environment, Figure 3. colloidal stability mpEG-pla, Gft@nps, Dox@nps, and Dox@Gft-nps for 24 hrs with an aqueous solution and DmEm cell culture medium were investigated by Dls methods.Data are presented as mean ± sD (n = 3).
respectively.As demonstrated in Figure 4, the release of DOX at pH 5.5 was faster than that at pH 7.4.This could be ascribed to the improved hydrophilicity of DOX and the weakened electrostatic interactions among carboxyl groups in mPEG-PLA and the amine group in DOX under acidic conditions.Additionally, DOX@GFT-NPs presented the maximum release of DOX at pH 7.4 comprising 5 mM GSH than at pH 7.4 or 5.5.The cleavage of disulfide bonds could describe this in the PLA backbone, and the resulting degeneration of DOX@GFT-NPs is stimulated by decreasing GSH.This result also showed that DOX@GFT-NPs were more susceptible to diminution than acidic pH.Likewise, the release of GFT at pH 5.5 was higher than at 7.4 in the absence of GSH.The GFT release from DOX@GFT-NPs improved the existence of GSH and was assessed more in the control groups (Figure 4).These outcomes suggested that DOX@GFT-NPs efficiently inhibited drug release in the extracellular environment but mediated reduction/pH dual responsive release of GFT and DOX in cancer cells [52].

Cellular internalizations of DOX@GFT-NPs
CLSM images and flow cytometric methods were used to detect free DOX fluorescence and investigate DOX@GFT-NPs cellular internalization.Next, cells were treated with DOX and DOX@GFT-NPs for 2 or 6 hrs, and FCA and CLSM examined SKOV3 ovarian cells.Figure 5A reveals that after a 2-hrs incubation, the fluorescence intensities of DOX@GFT-NPs were more extensive than PBS-treated, suggesting that SKOV3 cells successfully absorbed DOX@GFT-NPs.Since DOX dispersed into tumor cells more rapidly than DOX@GFT-NPs, which reached cells via the lengthier endocytosis route, the fluorescence intensity of the DOX-treated group was likely higher than that of the DOX@GFT-NPs-treated group.Extending the incubation periods from 2 to 6 hrs considerably enhanced the fluorescence intensities of DOX and DOX@GFT-NPs in the treated group, implying that both formulations showed time-dependent cellular internalization (Figure 5B).
CLSM examined into DOX@GFT-NPs' cellular absorption to determine if they could produce DOX's distinctive red fluorescence.HOECHST 33342 gave the nuclei of the cell a bright-blue color.Figure 6 illustrates the DOX@GFT-NPs and DOX released into the nuclei upon intracellular responsive DOX release were successfully absorbed by SKOV3 cells.The red fluorescence of DOX and DOX@GFT-NPs treated groups resembled the blue fluorescence color of the cell nuclei.The FCA findings show that the fluorescence intensity of DOX@GFT-NPs treated cells rises after 2 to 6 h of incubating.Our outcomes demonstrated that cancer cells effectively released and absorbed the coloaded anticancer drugs from DOX@GFT-NPs.

In vitro synergistic anticancer effects
In vitro, the synergistic anticancer effects of DOX@GFT-NPs were investigated by treating SKOV3 cells for 48 or 72 hrs with GFT-free, DOX-free, GFT@NPs, DOX@NPs, and DOX@GFT-NPs.The MTT analysis was performed to determine the viability of the SKOV3 cells.The viability of cells in NP-treated samples was minimally harmed (Figure 7).On the other hand, cytotoxicity against SKOV3 cells with dose-responsive for all drug formulations, as shown in Figure 7. GFT@NPs and DOX@NPs destroyed  tumor cells more efficiently than GFT-free and DOX-free.GFT-free and DOX-free may have dispersed into ovarian cancer cells, which might be the source of this event.DOX@ NPs and GFT@NPs, on the other hand, utilized the endocytic pathways to enter the cancer cells.The outcomes of the drug release of nanoparticle drugs took extended than estimated.DOX formulations had a similar level of cytotoxicity as GFT-containing formulations.The group treatment with DOX@GFT-NPs saw a more significant reduction in cell viability than the GFT@NPs or DOX@NPs groups.DOX emitted from nanoparticles creates a GFT@NPs synergy (Figure 8).

Morphological investigation of SKOV3 cells
The acridine orange/ethidium bromide differential staining approach examined apoptosis-induced morphological alterations by GFT-free, DOX-free, GFT@NPs, DOX@ NPs, and DOX@GFT-NPs.Viable (light green), early and late apoptotic, and nonviable cells (red colored) were defined in the stained cells [53][54][55].Condensed nuclei, membrane flaking, and apoptotic bodies were seen in cells treated with DOX@GFT-NPs (Figure 9A).In contrast, the nuclear architecture of the control cells was intact.However, a few apoptotic bodies were observed in SKOV3 cells treated with GFT-free, DOX-free,  GFT@NPs, DOX@NPs, and DOX@GFT-NPs for 24 h of acceptable IC 50 concentration.The apoptotic mode of cell death percentage ratio is shown in Figure 9B.
The HOECHST 33342 staining methods established that GFT-free, DOX-free, GFT@ NPs, DOX@NPs, and DOX@GFT-NPs-induced apoptotic cell death was linked with nuclear condensation and fragmentation [56][57][58].In this examination, SKOV3 cells were stained with HOECHST 33342 and evaluated under a fluorescence microscope after  being exposed to DOX@GFT-NPs for 24 hrs at acceptable IC 50 concentration.HOECHST 33342 is a DNA intercalating agent that binds to the DNA between the nucleotides.An increase in the number of positive cells.Figure 10A showed that DOX@GFT-NPs triggered nuclear alterations in cells that were in the process of dying.The fragmented and condensed nucleus ratios are shown in Figure 10B.

Figure 7 .
Figure 7. the cell viability of the sKoV3 cells was examined by mtt assay.cell viability of sKoV3 cells incubated with mpEG-pla for 48 hrs and 72 hrs at 37 °c.Data are presented as mean ± sD (n = 3).