One Pot Doxorubicin Partitioning and Encapsulation on Silica Nanoparticle, Applying Aqueous Two Phase System for Preparation of pH-Responsive Nanocarriers

Providing an efficient system for drug delivery and chemotherapy has always been an important issue. Modification of the surface of silica nanoparticles (SiO 2 ) provides an opportunity for achieving stimulus-sensitive drug delivery system. Here, we have modified the surface of SiO 2 using hydrogen bonding interactions by employing an aqueous two-phase system (ATPS) based on polyethylene glycol and lysine. This novel biocompatible ATPS provides an environment for simultaneous drug encapsulation, SiO 2 modification, and drug partitioning in one pot. Addition of SiO 2 to ATPS increased the partitioning of doxorubicin (DOX) as an anti-cancer drug from 47.92 in the absence of nanoparticles to 92.33 due to the interactions between drug and nanoparticles. The formation of nanoformulation and its characteristics were investigated applying microscopy, spectroscopy and thermal analysis. Drug release study demonstrated that DOX is loaded on nanoformulations efficiently with an encapsulation efficiency of 63.84% and shows lower release in physiological environment compared to the unmodified nanoparticles. While in acidic conditions of pH 5.5, significant increase was observed in the release profile. MTT assay on MCF-7 cancer cells confirmed that the nanoformulations were non-toxic and DOX-loaded nanocarrier showed anti-cancer behavior. These results indicate that the prepared nanoformulations are promising nanocarriers for controlled drug release purposes.


Introduction
Cancer has long been a major threat to human health, and despite many efforts and studies, it has always been a significant challenge 1,2 . Chemotherapy, as the most widely used method for cancer treatment, destroys both cancerous and normal cells and causes severe side effects 3 . On the other hand, a small portion of the introduced drug is delivered to the tumor tissues diminishing the efficiency of chemotherapy 4 . This causes the drug to be prescribed in higher doses, which leads to the systematic removal of drug from the body and is not affordable 5,6 . Drug resistance of cancers to chemotherapy is another factor that results in treatment failure 7 . To address these issues, it is necessary to design targeted drug delivery systems in order to: 1) target the tumor site and decrease the drug concentration in other tissues; 2) release the drug in response to internal or external stimuli such as redox, pH, biological molecules in the tumor environment, magnetic field, light and temperature [8][9][10][11][12] . Targeted nano-drug delivery systems with small sizes (10 to 100 nm) enhance permeation through the newborn blood vessels of tumor tissues with slower clearance due to the lack of lymph 12 . These drug nanocarriers including micelles 13,14 , polymersomes 15 , liposomes 16 , dendrimers 17 , carbon nanotubes 18 , magnetic nanoparticles 19 and silica nanoparticles 20,21 are of high importance because of their higher permeability, detection of target cells, accumulation at cancer sites, less side effects, and more efficient treatment 4,5,22 .
Modification of the surface of silica nanoparticles offers opportunities to prevent nanoparticles accumulation and elimination by proteins and ions in the physiological microenvironment and barricade the burst release of the drug, which increases the level of the drug in normal tissues and reduces its concentration at the tumor site 34,40,41 . A well-investigated method to modify the surface of nanoparticles is PEGylation 42,43 . Polyethylene glycol (PEG) is an FDA approved non-toxic, non-immunogenic, and non-antigenic polymer 34,44 , which forms a hydrophilic layer around the nanoparticle and increases the water solubility, and improves dispersion and colloidal stability using the stealthing effect of PEG through rapid movement of hydrated polymer chains 23,40,45,46 .
PEGylation can significantly prevent protein adsorption on nanoparticle surface and rapid clearance of nanoparticles by RES that leads to increased EPR and circulation time 12 .
Polymeric compounds are attached on the surface of inorganic nanocarriers in various methods, including strong chemical bonds (covalent or ion-covalent bonds), the reaction of endfunctionalized-polymers with the functional groups on nanoparticles, initiation of polymerization from the surface, and physical interactions such as hydrogen bonding and Van der Waals interactions 23,47,48 . Non-covalent bonds are more sensitive to stimulus. Among these, hydrogen bonding is a selective and relatively potent interaction that occurs only between the hydrogen bond donor and receptor and is very sensitive to pH changes 49,50 . This bond has low activation energy and can occur at room temperature. The surface of silica nanoparticles is covered by silanol, which is prone to form hydrogen bondings with PEG and drugs 49,51 . So far, a considerable amount of effort has been devoted to designing pH-sensitive polymeric nanocarriers using hydrogen bonding 49,50,52,53 . Hydrogen-bonded carriers can be used to deliver drugs to tumors that have an acidic environment. In addition, hydrogen bonding between the carrier and the drug can also prevent the immediate release of drug 49 .
Another compound applied in surface modification of nanoparticles is lysine, owing to its low cost, high compatibility, and availability [54][55][56][57] . Lysine is an essential compound in the body for strengthening the calcium absorption from the gastrointestinal tract, helping bone growth, collagen and antibody production, and tissue repair. Lysine is one of the simplest essential amino acids with one amine group in its side chain prone to interact with the hydroxyl groups of the silica surface through hydrogen bonding [55][56][57] .
Aqueous two-phase systems (ATPS) consist of an aqueous mixture of two water-soluble components 58 that are incompatible with each other and can split into two aqueous phases above a critical concentration 59,60 . This capability offers a tool for a variety of applications, including the isolation of sensitive biomolecules such as cells, enzymes, nucleic acids and proteins, encapsulation, enrichment, and delivery of active compounds and mimicry of cellular environment 17,[60][61][62][63][64] . Due to water presence in both phases and low interfacial tension between the phases, ATPSs provide a compatible medium for biomolecules 60,65,66 .
A tremendous effort has been made on PEG-based ATPSs 67,68 . However, the formation of ATPS using amino acids is less investigated. Amino acids are inner salts with a high affinity for water, and despite their weaker soluting-out ability compared to inorganic salts, they can form a benign ATPS for biological materials 60,61 . Therefore, in this research, a novel biocompatible PEG-lysine based ATPS is presented. This ATPS, as the first example of it, is employed for simultaneous partitioning and loading of doxorubicin (DOX) on silica nanoparticles, which can be modified in the presence of PEG and lysine ( Figure 1). Doxorubicin is one of the most widely used drugs in treating of various cancers, which disrupts cancer cell proliferation. Loading DOX on a targeted 6 carrier can help to reduce its side effects on normal cells and increase its effect on cancer cells 34 .
The hydroxyl, amine, and phenolic groups of doxorubicin can bound to silanol on the surface of silica through hydrogen bondings 69,70 , and in acidic condition, these bonds become unstable and protonated, leading to the release of DOX. Therefore, the phase equilibrium between PEG and lysine in an aqueous medium was investigated, and then using this ATPS, the partitioning of DOX in the absence and presence of silica nanoparticles was investigated. The release of DOX from nanocarriers resulting from self-assembly of the components was studied and its toxicity effect on MCF-7 breast cancer cells was evaluated.

Binodal curve and tie lines
As shown in Figure 2, PEG6000 and lysine were able to form an aqueous two-phase system and the liquid-liquid equilibrium diagram was determined at 298 K and atmospheric pressure. Since amino acids are weaker soluting-out induced species 60 , the two-phase region is formed at higher lysine concentrations, but still represents a large immiscibility region. The Merchuk parameters were obtained by fitting the experimental binodal data (Table 1) to employ in the determination of each phase composition at different mixture points.
Various mixture points (MP) were chosen to study the partitioning of DOX in PEG6000/lysine ATPS. The composition of each phase was obtained using the gravimetric method, mass balance, and the Merchuk equation (1-5), which are presented in Figure 2 and Table 2. As can be seen, the top phase is rich in PEG and lysine is dominant in the bottom phase. By increasing PEG wt%, the immiscibility increases, and at constant lysine wt%, each phase gets richer in its main component, which leads to higher TLL. Increasing the lysine wt% improves the phase separation as well, due to the more soluting-out effect at higher lysine concentrations. Comparing the mixture point of highest lysine wt% with highest PEG wt% and their phase compositions indicate that PEG wt% has more impact on phase separation, since the increased immiscibility resulted from higher PEG concentration is more considerable compared to the slightly increased soluting-out effect resulted from higher lysine concentration 60 .

Partitioning of DOX in PEG-lysine ATPS
The partitioning of DOX was investigated in the studied mixture points and is reported in Table 3.

FTIR analysis
The obtained nanoformulation, as well as all the components of the top phase, were analyzed through FTIR spectroscopy to investigate the effect of SiO2, and the resulting spectrum is presented in Figure 3. In the bare SiO2 spectrum, before water exposure, the peaks at 473, 798,

TGA analysis
Thermal decomposition of the obtained nanoformulation was carried out using thermal gravimetric analysis (TGA) at 25 to 600℃. Figure 4 shows the TGA thermograms of pure DOX, SiO2, PEG, lysine and DOX@nanoformulation. The 26% mass reduction of SiO2 around 100℃ is due to physically absorbed water evaporation. The nanoformulation TGA thermogram shows a weight loss around 100℃ related to water residuals. The second weight reduction occurred at 306℃. This weight loss could be attributed to lysine presence in the top phase, which has formed hydrogen bonding to the SiOH on the silica nanoparticle surface according to the decomposition temperature range of pure lysine, which occurs at a wide temperature range (starting at about 289℃, Figure 4).
The results is in accordance with the reported TGA analysis of lysine 79,91 , and lysine-SiO2 conjugates 56,57 . The subsequent decomposition appears at 327-484℃ due to decomposition of PEG, which starts at higher temperatures compared to pure PEG (201℃-510℃, Figure 4) 34 . The higher decomposition temperature of the DOX@nanoformulation thermogram confirms the formation of hydrogen bonding, which increases the thermal stability of the nanoformulation 92-94 .

Characterization of DOX@nanoformulation
To further analyze the properties of the nanoformulations, the hydrodynamic diameter was     Figure 8 (0 h) shows the absorbance of DOX@nanoformulation indicating that DOX was successfully loaded on the nanoformulations. As reported in Table 5, the drug encapsulation efficiency (DE%) and loading capacity (LC%) were measured by UV-Vis spectroscopy and calculated by equations (11) and (12). Surface modification has increased the drug encapsulation efficiency significantly compared to DOX@SiO2 nanoparticles. The obtained DE% is in the same range as reported chemically grafted PEG-mesoporous SiO2 nanoparticles 34    Following the experiments confirming the formation of PEGylated drug carrier, the DOX@SiO2 and DOX@nanoformulation were evaluated for their release kinetics, as represented in Figure 9.

Drug loading and release study
To ascertain that the drug carrier has limited the diffusion of DOX out of the structure, free drug release was also investigated. As shown, more than 99% of the free DOX was released within the first 4 hours. Compared to the free DOX, the release profile of the nanocarriers was considerably sustained at physiological pH. 35% of the drug was released at the first 5h confirming successful drug loading on PEGylated SiO2. The decrease in the absorbance of DOX in the nanocarriers solution is related to DOX concentration decrease, which confirms the DOX release ( Figure 8).
60% of DOX was released after 72h at physiological pH, and 40% remained in the carrier, suggesting the sustained and stable nanoparticle structure at pH=7.4 and good compatibility of DOX with the carrier. The release profile of DOX from bared SiO2 nanoparticles at physiological pH was studied to investigate the effect of modification on drug release. As can be seen in Figure   9, more than 76% of DOX was released from SiO2, indicating PEG prevention of the drug release in modified nanoparticles.
We were intrigued to study the behavior of DOX@nanoformulation at pH 5.5, which imitates the tumor environment and can affect hydrogen bonding. As can be seen in Figure 9, in acidic condition, a fast release up to 57% was obtained within 5h and 73% of the drug was released after 72h. The release profiles show that the DOX release from modified nanoparticles is pH-dependent.
The acidic conditions can break hydrogen bonding by protonation of NH2 groups on DOX and the hydroxyl groups on SiO2 surface. In addition, an acidic environment can increase the hydrophilicity of DOX, which improves its solubility 70,104 . The hydrogen bonding break and increased solubility of DOX led to the increased drug release from the carrier in the acidic pH.
Comparing the outcomes to the literature 34,105,106 indicates that the DOX@nanoformulation can retain the drug encapsulated in pH 7.4 and release the drug in acidic environment more efficiently, suggesting an effective pH-sensitive drug nanocarriers for DOX delivery to cancerous tissue.

Cytotoxicity assay by MTT
The cytotoxicity of the nanoformulations was evaluated by MTT assay. MCF-7 cells were treated with different concentrations of DOX, nanoformulation, and DOX@nanoformulation for 48h. The nanoformulation revealed to be non-toxic. As shown in Figure 10, the cell viability was 76.09% of control even up to a concentration of 1000μg/mL, confirming that the prepared carrier is biocompatible and safe for drug delivery applications. DOX@nanoformulation showed cytotoxicity at concentrations higher than 62.5μg/mL, which carries 0.5μg/mL DOX. Considering the partial release of DOX from nanoformulations after 48h at pH=7.4, almost the same toxicity as free DOX was obtained. This observation proves that the DOX activity is retained after drug loading. The viability of MCF-7 cells decreased as the concentration of the carrier increased.
However, the viability could be affected by the slow release of DOX from the carrier at the physiological pH.

Materials and instruments
Polyethylene

Phase diagram and tie lines
The binodal curves were determined through cloud point titration method as explained in literature 59 (5) Where the subscripts Top and Bot refer to the top and bottom phases, respectively. The tie line length and the slope of the tie lines were calculated using equations (6) and (7), respectively.

Partitioning of doxorubicin
Partitioning of doxorubicin was investigated through an established procedure 59,110

Partitioning of DOX in the presence of SiO2 and drug loading
The same procedure was applied to investigate the partitioning of doxorubicin in the presence of SiO2. Briefly, an aqueous solution of SiO2 (200mg/mL) was sonicated for 1h. The drug was added to the solution and it was sonicated for 30 more minutes. The ATPS mixture points were prepared by adding the desired amount of PEG and lysine and the solutions were stirred vigorously. The ratio of SiO2/DOX was kept at 10 in all samples. As before, to measure the partitioning of the DOX in the presence of SiO2, the two phases were separated carefully, and the DOX absorbance was measured by UV-Vis spectrophotometer.

Characterization of nanoformulations
In order to separate the drug-loaded PEG/lysine coated SiO2 nanoformulation, the top phase was centrifuged at 10000rpm for 30min and the supernatant was decanted. The sediment was dried under vacuum and characterized and analyzed using TGA and FTIR. The size of nanoformulations was measured in aqueous solutions by DLS and the morphology was determined through microscopic observation methods of TEM and AFM. The drug encapsulation efficiency (DE%) and loading capacity (LC%) were obtained using UV-Vis spectroscopy measurement and the following equations.

Drug release
The dialysis tubing method was employed to study the drug release profiles with three replicates 13 . 20 mg of DOX@nanoformulation or DOX@SiO2 was dissolved in 2ml DIW and placed in 6 kDa MWCO dialysis tubing and dialyzed against 140mL saline phosphate buffer (PBS) and 1% v/v Tween 80 with pH = 7.4 and 5.5. At specified time intervals, 20 μL samples were taken from dialysis tubing. The samples were diluted and the unreleased drug concentration was measured by UV-Vis spectroscopy.

Cell proliferation assay
MTT assay was employed to study the cytotoxic effect of free drug, blank nanocarriers, and drugloaded nanocarrier 111,112 . 10 4 cells of MCF-7 were cultured on each well of 96-well plates and were incubated for 24h. Afterward, the medium was replaced with mediums containing 100 μL of DOX, nanoformulation, and DOX@nanoformulation at various concentrations. After 48h of incubation, the medium was removed carefully and 20 μL of MTT solution in PBS (5mg/mL) was added. The plate was incubated for 4 h at 37℃ in darkness. Upon completion, 100 μL of DMSO was added to each well and the plate was shaken for 20min to dissolve the formed formazan crystals through the metabolic activity of mitochondria of live cells. The salts concentration quantification was performed using spectrophotometry analysis and measuring the absorbance of the samples at 570 nm and 690 nm (as the reference wavelength). All experiments were performed in three replicates.

Conclusion
The possibility of developing a method for simultaneous nanocarrier preparation and drug encapsulation can notably influence the practicability, efficacy, and accessibility of drug carriers.
We employed a new aqueous two-phase system based on polyethylene glycol and lysine (as an essential amino acid in the human body) to investigate the partitioning of DOX, which is an important parameter in purification processes. The addition of silica nanoparticles to the ATPS increased the doxorubicin partitioning considerably, suggesting strong interactions in the system.
Analyzing the obtained assemblies showed that due to the formation of hydrogen bonding between the components in the system, including DOX, SiO2, PEG, and lysine, which are all prone to form this non-covalent bonding, drug loading and SiO2 surface modification can occur at the same time.
The formed biocompatible nanocarrier offers an encapsulation efficiency of 63.84%. Investigation of the release profile of the nanocarriers showed that the surface modification prevented the drug escape and showed a more stable release compared to the bare DOX-loaded SiO2. Evaluation of drug release in acidic conditions indicated that the hydrogen bondings between components were pH-responsive and accelerated drug release. Evaluation of the toxicity of this carrier on MCF-7 breast cancer cells demonstrated that the nanocarriers had no cytoxicity and encapsulation of the drug showed high anti-cancer efficacy. The obtained results suggest that a biocompatible ATPS can be applied for simultaneous drug partitioning and loading and is a promising method for drug delivery purposes.