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

TL;DR: Results indicate that the prepared nanoformulations were non-toxic and DOX-loaded nanocarrier showed anti-cancer behavior and are promising Nanocarriers for controlled drug release purposes.

Abstract: Providing an efficient system for drug delivery and chemotherapy has always been an important issue. Modification of the surface of silica nanoparticles (SiO2) provides an opportunity for achieving stimulus-sensitive drug delivery system. Here, we have modified the surface of SiO2 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, SiO2 modification, and drug partitioning in one pot. Addition of SiO2 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.

Summary

1 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 .
• 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 .
• 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 .
• Therefore, in this research, a novel biocompatible PEG-lysine based ATPS is presented.
• Doxorubicin is one of the most widely used drugs in treating of various cancers, which disrupts cancer cell proliferation.

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.
• 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

• DOX, as a hydrophobic biomolecule (logP=1.41) 71 is inclined to partition to the top phase, which is PEG-rich and possesses lower water content and hence is less hydrophilic compared to the lysine phase with higher hydrophilicity (logP= -3.8).
• Higher lysine wt% at constant PEG wt% in the feed improves the TLL and decreases K because of the lower water concentration in the bottom phase.
• According to the obtained results, higher lysine and lower PEG concentrations adversely affect DOX partitioning.
• The effect of SiO2 NP on DOX partitioning SiO2 NP was added to the ATPS mixture points with the highest and lowest DOX partition coefficients to investigate its effect on partitioning.

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 .
• The SiO2 surface contains SiOH at room temperature, and it can be effectively hydroxylated in the presence of water and can form hydrogen bonding 51, 56, 78 .
• Due to the low activation energy of hydrogen bonding, it can occur at room temperature.
• FTIR analysis of pure DOX, SiO2, PEG, lysine and DOX@nanoformulation.

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 nanoformulation TGA thermogram shows a weight loss around 100℃ related to water residuals.
• The second weight reduction occurred at 306℃.

Characterization of DOX@nanoformulation

• To further analyze the properties of the nanoformulations, the hydrodynamic diameter was measured by dynamic light scattering.
• TEM micrograph of the nanoparticles and TEM size distribution are shown in Figure 6 .
• An average diameter of 25.66 nm was obtained from TEM for DOX@nanoformulations.
• The phase image of AFM demonstrates that the surface of silica nanoparticles was modified according to the presence of different phases, which indicates the presence of different materials 100, 101 .
• It can be observed that the nanoparticles are dispersed and do not form agglomerations.

Drug loading and release study

• 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).
• 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.

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.
• DOX@nanoformulation showed cytotoxicity at concentrations higher than 62.5μg/mL, which carries 0.5μg/mL DOX.
• 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.

Materials and instruments

• Thermal analysis of the samples was explored through thermal gravimetric analysis with a heating rate of 10°C/min (TGA, TGA-50, Shimadzu).
• A UV-Vis Spectrophotometer (DR3900, HACH, USA) was applied to determine the drug concentration.
• Aqueous solutions of SiO2 nanoparticle were sonicated using an ultrasonic bath (FALC, Italy).
• SZ-100z dynamic light scattering (DLS) analyzer (Horiba Jobin Jyovin) was used to measure the hydrodynamic diameter.
• Transmission electron microscopy (TEM) studies were performed with a Philips EM 208S instrument at a voltage of 100 kV.

Phase diagram and tie lines

• The binodal curves were determined through cloud point titration method as explained in literature 59, 107 at 298K and atmospheric pressure.
• The PEG solution was added dropwise to the lysine solution until the appearance of turbidity leading to the first point of binodal curve, which indicates the beginning of phase separation.
• The turbidity is removed by adding water and the procedure is repeated.
• The Merchuk equation 108 was applied to correlate the obtained binodal data.
• 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 .
• Briefly, ATPSs composed of various concentrations of PEG, lysine, and deionized water (DIW) containing 0.05mg of doxorubicin were prepared gravimetrically (u(m)=10 -4 g) at 298K and atmospheric pressure.
• The phases were separated intently, and the mass and volume of each phase were measured.
• The drug concentration in each phase was obtained by analyzing samples of each phase using the calibration curve and UV-Vis analysis at 481nm.
• Since the samples were diluted by DIW and other components do not show absorption at visible wavelengths, DIW was used as blank, and all experiments were performed with three replications.

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.
• 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 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.

4 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.
• The authors 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%.

Figures

Table 1. The Merchuk parameters.

Figure 1. Schematic representation of nanocarrier formation and drug loading in PEG/lysine-based aqueous two-phase system.

Table 4. Partition coefficient of DOX and EE% in the presence of SiO2 NP.

Figure 6. Transmittance electron microscopy micrograph of DOX@nanoformulation and its corresponding size histogram.

Figure 7. A) 2D and B) 3D AFM height and C) phase images of DOX@nanoformulation and D) AFM size histogram.

Table 3. Partition coefficient of DOX, Vr and EE%

Figure 5. DLS size distribution of DOX@nanoformulation.

Figure 9. In vitro release profile of DOX from nanocarriers at pH 7.4 and 5.5.

Figure 4. TGA analysis of pure DOX, SiO2, PEG, lysine and DOX@nanoformulation.

Figure 10. MTT assay of MCF-7 cell viability after treating with A) DOX, B) nanoformulation, and DOX@nanoformulation for 48h at various concentrations.

Table 5. Drug encapsulation efficiency (DE%) and loading capacity (LC%) of DOX-loaded nanocarriers.

Figure 3. FTIR analysis of pure DOX, SiO2, PEG, lysine and DOX@nanoformulation.

Table 2. Partition coefficients of DOX and weight fraction of components in PEG6000-lysine ATPS

Figure 2. The phase diagram and studied mixture points (MP) and their corresponding tie lines.

Figure 8. The absorbance of aqueous solution of DOX@nanoformulation at 0, 5 and 24 hours of dialysis in PBS.

Full text

One Pot Doxorubicin Partitioning and Encapsulation
on Silica Nanoparticle, Applying Aqueous Two
Phase System for Preparation of pH-Responsive
Nanocarriers
Mojhdeh Baghbanbashi
Amirkabir University of Technology (Tehran Polytechnic)
Gholamreza Pazuki (  ghpazuki@aut.ac.ir )
Amirkabir University of Technology (Tehran Polytechnic)
Sepideh Khoee
University of Tehran
Research Article
Keywords: Controlled release, pH-sensitive, Aqueous two-phase system, Hydrogen bonding, Silica
nanoparticle, Lysine
Posted Date: July 21st, 2021
DOI: https://doi.org/10.21203/rs.3.rs-732929/v1
License:   This work is licensed under a Creative Commons Attribution 4.0 International License. 
Read Full License

1
One Pot Doxorubicin Partitioning and Encapsulation on Silica
Nanoparticle, Applying Aqueous Two Phase System for Preparation
of pH-Responsive Nanocarriers
Mojhdeh Baghbanbashi
1
, Gholamreza Pazuki
1,*
, Sepideh Khoee
2
1
Department of Chemical Engineering, Amirkabir University of Technology (Tehran
Polytechnic), Tehran, Iran.
2
School of Chemistry, College of Science, University of Tehran, Tehran, Iran.
1
,* Corresponding author: Tel: +98-021-64543159. Fax: +98-021-66405847. E-mail address:
ghpazuki@aut.ac.ir (G.R. Pazuki)

2
Abstract
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.
Keywords: Controlled release, pH-sensitive, Aqueous two-phase system, Hydrogen bonding,
Silica nanoparticle, Lysine.

3
1 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
8-12
. 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
.
Silica nanoparticles (SiO
2
NP) provide many advantages, including high stability
23
,
biocompatibility
24,25
, tumor site accumulation ability through enhanced permeability and retention
(EPR) effect
26
, low toxicity
27-32
, simple synthesis and surface modification, uniform and
adjustable morphology, easy and low-cost large-scale synthesis
33
and high surface area to volume
ratio
28
. The application of SiO
2
NP, which is approved by food and drug administration (FDA) as

4
safe materials
34
, have been widely studied in the diagnosis and treatment of diseases as imaging
agents, biosensors, and drug and gene carriers
21,25,35-39
.
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 end-
functionalized-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

Frequently asked questions

Q1. What are the contributions in "One pot doxorubicin partitioning and encapsulation on silica nanoparticle, applying aqueous two phase system for preparation of ph-responsive nanocarriers" ?

In this paper, the authors proposed a targeted drug delivery system to target the tumor site and decrease the drug concentration in other tissues. 

Q2. What are the future works mentioned in the paper "One pot doxorubicin partitioning and encapsulation on silica nanoparticle, applying aqueous two phase system for preparation of ph-responsive nanocarriers" ?

The possibility of developing a method for simultaneous nanocarrier preparation and drug encapsulation can notably influence the practicability, efficacy, and accessibility of drug carriers. The addition of silica nanoparticles to the ATPS increased the doxorubicin partitioning considerably, suggesting strong interactions in the system. 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. 

Q3. What are the advantages of silica nanoparticles?

Silica nanoparticles (SiO2 NP) provide many advantages, including high stability 23, biocompatibility 24,25, tumor site accumulation ability through enhanced permeability and retention (EPR) effect 26, low toxicity 27-32, simple synthesis and surface modification, uniform and adjustable morphology, easy and low-cost large-scale synthesis 33 and high surface area to volume ratio 28. 

Q4. What was the method used to study the cytotoxic effect of free drug?

Cell proliferation assayMTT assay was employed to study the cytotoxic effect of free drug, blank nanocarriers, and drugloaded nanocarrier 111,112. 

Q5. What is the effect of SiO2 on DOX partitioning?

The SiO2 surface contains SiOH at room temperature, and it can be effectively hydroxylated in the presence of water and can form hydrogen bonding 51,56,78. 

Q6. What is the common compound used in surface modification of nanoparticles?

Another compound applied in surface modification of nanoparticles is lysine, owing to its low cost, high compatibility, and availability 54-57. 

Q7. What is the effect of adsorption on the surface of silica nanoparticle?

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. 

Q8. How much SiO2 was added to ATPSs?

Since 0.5 mg of SiO2 (0.025 wt%) was added to ATPSs, the effect of the additive on binodal curve could be considered negligible 73-75. 

Q9. How was the decomposition of the nanoformulation performed?

TGA analysisThermal decomposition of the obtained nanoformulation was carried out using thermal gravimetric analysis (TGA) at 25 to 600℃. 

Q10. What was the method used to study the drug release profiles?

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 6kDa MWCO dialysis tubing and dialyzed against 140mL saline phosphate buffer (PBS) and 1% v/v Tween 80 with pH = 7.4 and 5.5. 

Q11. What is the effect of silica nanoparticles on the doxorubic?

The addition of silica nanoparticles to the ATPSincreased the doxorubicin partitioning considerably, suggesting strong interactions in the system. 

Q12. What is the effect of surface modification on the nanoparticles?

This can beattributed to surface modification, which makes the nanoparticles more stable by acting as a solvation layer and preventing interactions between SiO2 nanoparticles 83,102,103. 

Q13. What is the weight loss of lysine in the FTIR spectrum?

Thisweight loss could be attributed to lysine presence in the top phase, which has formed hydrogenbonding 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). 

Q14. What was the temperature of the samples?

Thermalanalysis of the samples was explored through thermal gravimetric analysis with a heating rate of10°C/min (TGA, TGA-50, Shimadzu). 

Q15. What is the role of the surface of silica nanoparticles in the drug delivery?

Modification of the surface of silica nanoparticles (SiO2) provides an opportunity forachieving stimulus-sensitive drug delivery system. 

Q16. What was the effect of the nanocarrier on the cancer cells?

Evaluation of the toxicity of this carrier on MCF-7breast cancer cells demonstrated that the nanocarriers had no cytoxicity and encapsulation of thedrug showed high anti-cancer efficacy. 

Q17. What was the method used to determine the binodal curves?

The binodal curves were determined through cloud point titration method as explained in literature 59,107 at 298K and atmospheric pressure. 

Q18. What is the effect of DOX on breast cancer cells?

The release of DOX fromnanocarriers resulting from self-assembly of the components was studied and its toxicity effect onMCF-7 breast cancer cells was evaluated. 

Q19. What is the effect of SiO2 NP on DOX partitioning?

This improvement in DOX partitioncoefficient could result from the increased interactions by SiO2 NP, which was investigatedthrough further analysis of the separated nanoformulations obtained from the top phase.