A Universal Approach to Render Nanomedicine with Biological

Subscriber access provided by Kaohsiung Medical University

A Universal Approach to Render Nanomedicine with Biological Identity Derived from Cell Membranes Jingyi Zhu, Mingkang Zhang, Diwei Zheng, Sheng Hong, Jun Feng, and Xian-Zheng Zhang Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b00242 • Publication Date (Web): 27 Mar 2018 Downloaded from http://pubs.acs.org on March 27, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

A Universal Approach to Render Nanomedicine with

Biological

Identity

Derived

from

Cell

Membranes Jingyi Zhu,1,2 Mingkang Zhang1, Diwei Zheng1, Sheng Hong1, Jun Feng*,1 and Xian-Zheng Zhang*,1 1

Key Laboratory of Biomedical Polymers of Ministry of Education & Department of Chemistry,

Wuhan University, Wuhan 430072, P. R. China 2

Department of Chemical and Biomolecular Engineering, National University of Singapore,

Singapore 117585, Singapore

KEYWORDS: top-down preparation, biomimetic nanoengineering, cell membrane, biological identity, tumor biotargeting

ACS Paragon Plus Environment

1

Biomacromolecules 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 34

ABSTRACT: Biomimetic nanoengineering built through integrating the specific cell membrane with artificially synthetic nanomedicines represents one of the most promising directions for the actualization of personalized therapy. To address the technical hurdle against the development of this biomimetic technology, the present report describes the in-depth exploration and optimization over each critical preparation step, including establishment of nanoparticlestabilized dispersion system, cargo loading, membrane coating and product isolation. Magnetic iron oxides nanoparticles loaded with DOX is used as a typical model for the coating with cancer cell membranes, providing compact DNP@CCCM nanostructure well characterized by various techniques. Furthermore, the feasibility of this optimized approach in constructing biomimetic membrane-coated nanomedicines has been validated on the basis of the remarkably improved biofunctions, such as the targetability, magnetic property, hemolysis risk, macrophage evasion, in vitro cytotoxicity, in vivo circulation duration and in vivo principal component analysis postinjection. We hope this study about the technique optimization would prompt the advance of the biomembrane-camouflaged nanoparticles as a newly emerging biomimetic technology.


2

Page 3 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

INTRODUCTION Nanoparticle aided drug delivery pattern, also termed nanomedicine, has been acknowledged as a technological revolution toward traditional chemotherapy.1,2 Though the research in this field grows with leaps and bounds, the clinical translation of nanomedicine paradigm still remains huge obstacles due to the inadequate understanding about the nano-bio interactions occurring in body.3,4 These obstacles mainly include the instability during the circulation,5 the easy recognition and clearance caused by their intruder nature,6 the poor target ability due to the nonspecific uptake by tissues.7,8 In attempts to circumvent these biological barriers, the past decade has witnessed the increasingly great effort from researchers in nanomedicine construction by mimicking natural transport conveyer (red blood cells, neutrophils, platelets, exosomes, virus, etc.) with regard to the composition, the structure, and the molecular mechanism responsible for the biological action.9-21 Among the reported biomimetic strategies, utilization of endogenously derived substances as building blocks for nanomedicine construction is undoubtedly more advantageous because it can directly share the inherent characters and functions with the biological parents, which is hardly accessible for synthetic materials. Nowadays, cell membranecamouflaged nanomedicines (CMNs) are being intensively studied by coating functional nanoparticles (NP, e.g. gold nanocage,22 iron oxides,23 mesoporous silicon,24 upconversion nanoparticles (UCNPs),25 metal-organic framework (MOF)26 and other polymer-based nanoparticles27,28) with the cell membranes extracted from various types of cells. In addition to the desired functionality belonging to the nanoparticle core (e.g., cargo loading, in vivo imaging, magnetic targeting, phototherapy, etc.), what is most fascinating about these CMNs is the biological identity imparted by cell membrane camouflage, leading to the highly improved biocompatibility, the prolonged blood circulation half-life, the disease-seeking ability, and the


3


Page 4 of 34

strong tendency of homing to the homologous tumors developed from the source cells.26,27,29-31 CMN research represented one of the most promising directions to advance the bench-to-bedside translation of personalized treatment in future. The typical ‘top-down’ approach has currently taken a leading position for cell membrane coating to nanoparticles,32 because this approach excludes tedious chemical synthesis and thus exerts negligible impairments on the components contained in biomembranes, the loaded cargoes, and the function of nanoparticles. However, such ‘top-down’ pathway is technically challenged by the susceptibility of nanoparticles to aggregation prior to the membrane coverage and the insufficient cohesion between the two components. This dilemma is presented more profoundly for inorganic nanoparticles because of their high surface energy, leading to even the precipitation occurring during the membrane coating process.33 Though the surface modification for hydrophilicity improvement could promote the stability of nanoparticles in aqueous mediums, it would conversely hinder the contact coverage of cell membranes. 34 Besides, top-down coverage of biomembranes would encounter the issue of difficult isolation and low yield. Therefore, the establishment of a universally effective method of CMNs preparation is urgently needed so to push forward the rapid development of this newly emerging nanotechnology. Our lab has recently been engaged in the CMNs research and focused on the self-homing of cancer cell membrane coated NPs to homologous tumors.35-39 To address the above-mentioned issues, this study has detailed our efforts in optimizing ‘top-down’ preparation approach over each critical step, including establishment of nanoparticle-stabilized dispersion system, cargo loading, membrane coating and product isolation. (Scheme 1) Magnetic iron oxides nanoparticles (MION) adsorbed with the drug cargo of doxorubicin (DOX) are herein used as a typical nanoparticle model for the coating with cancer cell membrane to prepare DNP@CCCM


4

Page 5 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

because magnetic assistance could help collect the products efficiently. We next demonstrate the success of the optimized approach by investigating the biofunction improvement regarding the targetability, magnetic contribution, cellular biocompatibility, macrophage evasion, in vivo circulation duration, in vitro cytotoxicity and principal component analysis post in vivo injection.

Scheme 1. Schematic illustration of preparation process of cancer cell membrane-cloaked DNP@CCCM by a ‘top-down’ approach including the step of establishment of dextranstabilized DNP dispersion system, cargo loading, membrane coating and product isolation.

MATERIALS AND METHODS


5


Page 6 of 34

Materials. Dextran (Mw~5.0 K Da, 4.0 W Da) and poly(ethyleneglycol) (PEG, Mw~2.0 W Da) were purchased from Aldrich Chemical Co. Ltd. Doxorubicin hydrochloride (DOX·HCl) was provided by Zhejiang Hisun Pharmaceutical Co., Ltd. (China). Membrane protein extraction kit and phenylmethanesulfonyl fluoride (PMSF) were purchased by Beyotime Institute of Biotechnology (China). All other chemicals were purchased from Shanghai Chemical Reagent Ltd. and used without any treatments. NuPAGENovex 4-12% Bis-Tris Gel, Dulbecco's modified Eagle's Medium (DMEM), fetal bovine serum (FBS), 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazoliumbromide(MTT), Hoechst 33342, and phosphate buffered saline (PBS) were purchased from Invitrogen Corp. (Carlsbad, CA, USA). Measurements. Transmission electron microscopy (TEM) was carried out on a JEOL JEM 100CXII instrument at an accelerating voltage of 80 kV. The hydrodynamic size and zeta potential were determined by dynamic light scattering (DLS) on a Malvern Zeta sizer Nano-ZS ZEN3600instrument. Confocal microscopy was performed on a confocal laser scanning microscope (CLSM) (Nikon C1-si TE2000, Japan) and recorded by EZ-C1 software. Flow cytometric assay was carried out by flow cytometry (BD FACS Aria™ III, USA). The cytotoxicity was measured at 570 nm using a microplate reader (Bio-Rad, Model 550, USA). 1H nuclear magnetic resonance (1H NMR) spectra were recorded on a Varian Unity 300 MHz spectrometer using D2O as the solvent with phenylsulfonic acid as an internal standard. Preparation of Cracked Cancer Cell Membrane (CCCM). Human cervix carcinoma (HeLa) cells were incubated in DMEM containing 10% FBS and 1% antibiotics (penicillinstreptomycin). Cells were cultured in 10-cm cell dishes, and detached using a cell scraper, and then isolated by centrifugation treatment at 700 g for 5 min. The cell pellets were resuspended in pre-cooled PBS (pH~7.4) and centrifuged once more at 700 g for 5 min. After that, the obtained


6

Page 7 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

cells were suspended in a hypotonic lysing buffer containing membrane protein extraction reagent and phenylmethanesulfonyl fluoride (PMSF), and incubated in ice-bath for 10-15 min according to the manufacturer’s instructions. Afterwards, the cells were cracked by a freeze-thaw method repeatedly for three times followed by centrifugation at 700 g for 10 min at 4 oC. The supernatant was carefully collected and subjected to further centrifugation at 14,000 g for 30 min to acquire the cell membrane fragments. The products were lyophilized, weighed and stored at 80 °C. The lyophilized membrane materials are rehydrated in PBS (pH~7.4) prior to use. CCCMs from other cell lines, such as the human squamous carcinoma cell lines developed at the University of Michigan (UM-SCC-7), were collected using the same treatment.35 Preparation of Magnetic Iron Oxide Nanoparticles (MION) and DOX-Loaded Nanoparticles (DNP). Citric acid modified magnetic iron oxide nanoparticles (MION) were prepared according to the Massart precipitation method.40 Briefly, 3.0 mL of NH3·H2O (25~28%) was added to 20.0 mL aqueous solution of FeCl2 (0.43 g) and FeCl3 (1.18 g), and stirred for 0.5 h (80 oC) under argon atmosphere. Then, 2.0 mL of citric acid (0.5 g) was introduced and reaction continued for 1.5 h at 95 oC. After the dispersion was cooled down to room temperature, MION was finally obtained by dialysis against deionized (DI) water using a 14 KD cut-off cellulose membrane. A dynamic stable dispersion system was established prior to drug loading. Two polymers, polyethyleneglycol (PEG, Mw~2.0 W Da) and dextran with two kinds of molecular weight (Mw~5.0 K Da and ~4.0 W Da) were introduced in hopes to stabilize the DNP solution system. The typical preparation procedure was described as follows. Under ultrasonic vibration, MION (0.4 mg/mL) dispersed in dextran solutions (100 and 200 mg/mL for Mw~5.0 K Da dextran solution, 50 and 100 mg/mL for Mw~4.0 W Da dextran solution) was added dropwise with a predetermined amount of DOX·HCI solution (2.0 mg/mL, pH ~6.5). After standing


7


Page 8 of 34

overnight, there appeared apparent precipitation in all solutions except for the case using ~4.0 W Da dextran with the concentration of 100 mg/mL. In the identical manner, it failed to establish a stable DNP solution when utilizing PEG (Mw~2.0 W Da) as the stabilizer at the concentrations of 50, 100, 150, 200 mg/mL respectively. Therefore, 100 mg/mL of Mw~4.0 W Da dextran was exploited as the optimal condition to provide the stable DNP solution system for the following CCCM coating. DOX loading towards MION was achieved by nanoprecipitation method.41 In brief, under ultrasonic vibration, a predetermined amount of DOX solution (2.0 mg/mL, pH ~6.5) was added dropwise into 100 mg/mL of MION (0.4 mg/mL) containing dextran solutions (Mw~4.0 W Da). Then, DNP was purified by magnetic separation. Study of pH Influence on the Stability of DOX Loading. The stability of dextran-stabilized DNP solution system was also examined under different pH conditions of 7.4, 9.3 and 10.8 according to the pKa values of citric acid (pKa1=3.13, pKa2=4.76, and pKa3=6.4).42 Dextran (Mw~4.0 W Da) with the concentration of 100 mg/mL was used here, and the pH was adjusted to be 7.4, 9.3, and 10.8 respectively before drug loading. The control group was performed by replacing dextran with DI water. Photos were collected after a period of standing. And also, the hydrodynamic diameter of DNP in dextran-stabilized solution was monitored with the addition of DOX under different pH conditions using DLS. DLS results indicated that dextran-stabilized DNP could be prepared under mild neutral condition, which would be beneficial to the following membrane coating as well. Preparation of Cancer Cell Membrane Coated DNP (DNP@CCCM). To prepare cancer cell membrane cloaked DNP (DNP@CCCM) NPs, a successive extrusion approach was established.35 Briefly, 2.0 mL of dextran-stabilized of DNP (0.5 mg/mL) was dropwise added with 1.0 mL of CCCM dispersion (1.0 mg/mL) in deionized water. After homogeneous mixing


8

Page 9 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

by vortex treatment, the mixture was transferred into syringe and successively extruded through 2.0 µm, 800.0 nm and 450.0 nm water-phase filters. The obtained DNP@CCCM was further purified by magnetic separation to remove the stabilizer of dextran and free CCCM. The encapsulation efficiency and drug loading content of DOX in DNP@CCCM were calculated to be 91.3% and 16.8% respectively, based on the UV-vis absorbance measured at 480 nm, using a standard calibration curve experimentally obtained. SDS-PAGE Protein Analysis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) method was used to examine protein retention in DNP@CCCM, according to our previous literature.43 The CCCM samples in lithium dodecyl sulfate (LDS) loading buffer were heated to 90 oC for 10 min, and 20 µL of sample was loaded into each well of a NuPAGENovex 4-12% Bis-Trisminigel, using 3-(N-morpholino) propane sulfonic acid (MOPS) sodium dodecyl sulfate (SDS) as running buffer (Invitrogen) in an XCellSureLock Electrophoresis System based on the manufacturer’s instructions. Protein staining was accomplished using Coomassie Blue (Invitrogen) and destained in water overnight before imaging. Stability Study. The stability of DNP@CCCM dispersion was studied in different media including DMEM, 10% FBS containing PBS buffer, and PBS~7.4 buffer (10 mM). At the given time intervals, these dispersions in centrifuge tube were photographed to visually reflect the variations over standing period. DNP without dextran was served as control. Meanwhile, the hydrodynamic diameter and the size distribution were monitored by DLS. Hemolysis Test. 10 mL of blood from healthy rabbit was collected into an anticoagulation tube and the fibrinogen was removed by stirring with glass rods. Then, 10 mL of normal saline (NS) solution was added into the treated blood samples. The erythrocyte pellets were collected


9


Page 10 of 34

after centrifugation at 1500 rpm for 15 min, and washed thrice with NS solution. A 2% erythrocyte standard dispersion was thus obtained by adding a certain amount of NS solution to the erythrocyte pellets. 0.5 mL of DNP@CCCM in NS solution with different concentrations (50, 100, 200 µg/mL) was placed in a tube loaded with 0.5 mL of 2% erythrocyte standard dispersion. The DNP NPs with same concentration were served as control. The exposure of erythrocyte pellets to NS solution and distilled water was used as negative and positive control, respectively. The mixtures in all tubes were gently shook and incubated at 37 oC for 1 h prior to the centrifugation treatment at 1500 rpm for 5 min. Photos were taken and the hemoglobin in the supernatant was estimated on the basis of the absorbance recorded at 545 nm. The hemolysis rate was calculated using the mean optical density (OD) for each group as follows: hemolysis rate (%)=[OD(samples)-OD(negative

control)]/[OD(positive control)-OD(negative control)]×100%;

where OD(samples)

represents the OD value obtained in the presence of samples, OD(negative control) and OD(positive control) represents the negative and positive control mentioned above, respectively. Cell Culture. HeLa, UM-SCC-7, African green monkey kidney cell (COS7), and raw 264.7 macrophage cell were incubated in DMEM with 10% FBS and 1% antibiotics (penicillinstreptomycin, 10,000 U mL-1) at 37 oC in a humidified atmosphere containing 5% CO2. In Vitro Macrophage Uptake Study. Raw 264.7 macrophage cells were seeded at a density of 2.5×105 cells per single dish in 1 mL of DMEM containing 10% FBS and incubated at 37 oC for 24 h. The medium was then replaced with fresh medium containing DNP@HeLa CCCM (termed DNP@HeLa) or DNP ([DOX]: 1.5 µg/mL). After 2 hours incubation, the cells were rinsed with PBS, stained with Hoechst 33342 for 15 min, and finally photographed by confocal laser scanning microscopy (CLSM). For flow cytometric analysis (FCA), Raw 264.7 macrophage cells were seeded in 6-well plates at a density of 6×104 cells/well and cultured for


10

Page 11 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

24 h. After coincubation with DNP@HeLa or DNP ([DOX]: 1.5 µg/mL) for 2 h, the cells were washed thrice with PBS, detached by trypsin and collected by centrifugation treatment at 1000 rpm for 5 min. The collected cells were washed twice with PBS. The suspended cells were filtrated and examined by FCA. In Vivo Pharmacokinetic Study. For in vivo pharmacokinetic study, ten healthy mice were divided randomly into two groups and intravenously injected respectively with DNP@UM-SCC7 and DNP in PBS at a DOX dose of 2.5 mg/kg, respectively. 10 µL of the blood samples were taken at the time points of 5, 10, 30 min, 1, 2, 4, 6, 12, and 24 h post administration and then transferred into heparinized tubes. The samples were added with 0.4 mL of 0.075 N HCl and 0.4 mL of methanol, and then centrifuged at 10, 000 rpm for 10 min. The DOX content in supernatant was then measured according to the fluorescence absorbance intensity measured at 560 nm, using an experimentally obtained standard calibration curve. Total blood volume was estimated as 58.5 mL of blood per kg of body weight. In Vitro Homotypic Cell Targeting Studies. CLSM were used to investigate the homotypic targeting effect of DNP@CCCM NPs coated with UM-SCC-7 and HeLa cell membranes. In brief, UM-SCC-7 cells were seeded at a density of 2.5×105 cells per single dish in 1 mL of DMEM containing 10% FBS and incubated at 37 oC for 24 h. Then, the medium was replaced with the fresh medium containing DNP@UM-SCC-7 CCCM (termed DNP@UM-SCC-7) ([DOX]: 1.5 µg/mL). 2 h later, the cells were washed thrice with PBS, treated with Hoechst 33342 for 15 min, and finally photographed by CLSM and recorded by EZ-C1 software. The CLSM observation in COS7 cells was served as control. Under the identical conditions, CLSM observation was performed for DNP@HeLa NPs in HeLa and COS7 cell lines to further verify the homotypic targeting effect.


11


Page 12 of 34

In Vitro Cytotoxicity. The cell viability of DNP@UM-SCC-7 in UM-SCC-7 and COS7 cell lines was detected by MTT assay. Briefly, the cells were seeded in 96-well plates (6×103 cells/well) in 100 µL of DMEM containing 10% FBS for 24 h. The medium in each well was then replaced with 100 µL of fresh medium containing DNP@UM-SCC-7 with a series of concentrations. 2 h later, the medium was replaced with 200 µL of fresh medium and the culture was proceeded for another 48 h. Thereafter, 20 µL of MTT (5.0 mg/mL) was added into each well and further incubated for 4 h, and the medium was discarded and 150 µL of DMSO was added into each well to dissolve the formazane of MTT. Absorption at 570 nm was measured using a microplate reader (Bio-Red, Model 550, USA), and cell viability was calculated. Data were shown as mean ± standard deviation (SD) based on three independent measurements. In Vivo Metabolic Study. Female BALB/c nude mice (4-5 weeks old) were purchased from Zhongnan Hospital of Wuhan University (Wuhan, China) and fed with standard chow. All animals received care in compliance with the guidelines outlined in the Guide for the Care and Use of Laboratory Animals and the procedures were approved by the Wuhan University of China Animal Care and Use Committee. The mouse model bearing one UM-SCC-7 tumor on right hind limbs were established by subcutaneous injection of 1×106 UM-SCC-7 cells per mouse. When the tumor volume reached ~100 mm3, the mice were divided into six groups randomly (6 mice per group). The mice were intravenously injected with PBS, free DOX, DNP@UM-SCC-7, DNP@HeLa, and DNP@COS7 with a dose of 2.5 mg/kg DOX per mouse at the day of 1, 4, 7. Upon 12-day treatment, the mice were sacrificed. Preweighed UM-SCC-7 tumor tissues (200 mg) were homogenized using 1.0 mL of 50% acetonitrile/50% D2O. After centrifugation at 10,000 rpm for 10 min, the collected supernatant was evaporated to provide a solid product. The product was dissolved in 0.5 mL of D2O with phenylsulfonic acid as an internal standard prior to 1H


12

Page 13 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

NMR analysis. Following the 1H NMR spectral phase and baseline correction, each NMR spectrum was data-reduced to 256 regions of equal width (0.04 ppm) using the AMIX(Analysis of MIXtures) software package, version 2.0 (BrukerBiospin). Principal component analysis (PCA) was carried out using SIMCA-P 11.5 software (Umetrics AB, UMEÅ, Sweden) for multivariate data analyses. Statistical analysis was performed with SPSS statistical software package version 19.0 (SPSS Inc, Chicago, IL). Statistical Analysis. The data were expressed as mean ± SD on the basis of at least three independent experiments. Statistical analysis was performed using a Student's t-test. The differences were considered to be statistically significant for a p value

A Universal Approach to Render Nanomedicine with Biological

Recommend Documents