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| ORIGINAL ARTICLE |
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| Year : 2021 | Volume
: 5
| Issue : 4 | Page : 389-397 |
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Formulation of new intelligent nanoparticle inhibited H1N1 influenza subtype and SARS coronavirus type 2 (COVID-19) in vitro
Nima Shirmohammadi1, Arefeh Khodaee1, Maryam Rahimi2, Marzieh Vanayi1, Reza Aghanouri3
1 Department of Scientific Research, Kian Asa Center for Preventive Medicine (None Governmental Center Licensed by the Ministry of Health and Medical Education of Iran), SBMU Supervised Area, Tehran, Iran
2 Department of Biology, Faculty of Science, Malayer University, Malayer, Hamadan Province, Iran
3 Department of Scientific Research, Kian Asa Center for Preventive Medicine (None Governmental Center Licensed by the Ministry of Health and Medical Education of Iran), SBMU Supervised Area; Scientific Authority Center for Countering Biological Threats, Tehran, Iran
| Date of Submission | 01-Sep-2021 |
| Date of Acceptance | 12-Oct-2021 |
| Date of Web Publication | 14-Dec-2021 |
Correspondence Address:
Reza Aghanouri
Department of Scientific Research, Kian Asa Center for Preventive Medicine (None Governmental Center Licensed by the Ministry of Health and Medical Education of Iran), SBMU Supervised Area, Tehran, Iran; Scientific Authority Center for Countering Biological Threats, Tehran
Iran
 Source of Support: None, Conflict of Interest: None
DOI: 10.4103/bbrj.bbrj_265_21
Background: Rapid infection of the coronavirus and frequency of the subtypes are the main problems of drug and vaccine intervention during COVID-19 pandemic. New drug discovery to respond these needs, is the goal of study. Hence, considering structural and biological components of SARS-CoV-2, new intelligent particle designed and formulated and several dockings were done as in silico assay. Methods: Fe3O4 nanoparticles synthesized by the coprecipitation method, coated and functionalized. Chemical bindings (Fourier transform infrared assay), magnetic behavior (vibrating sample magnetometer assay), morphology (field emission scanning electron microscope assay), and cell toxicity and cell viability (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay) were checked and confirmed in vitro by exposure of particles to the H1N1 virus laboratory media exposure (BSL2 category). Results: Results show drug has ability to reduce more than 4 logarithms in the virus titration, in concentration of 70 μg/ml, so it was proved these nanoparticles could have antiviral effect. Although, because of lack of BSL3 standard laboratory, the antiviral effect on COVID-19 could not be performed on large scale. Conclusions: By the way, we concluded that new specific nanoparticles could make a new chance for COVID-19 drug therapy at any subtype exposure.
Keywords: COVID-19, nanoparticle, H1N1 influenza
How to cite this article:
Shirmohammadi N, Khodaee A, Rahimi M, Vanayi M, Aghanouri R. Formulation of new intelligent nanoparticle inhibited H1N1 influenza subtype and SARS coronavirus type 2 (COVID-19) in vitro. Biomed Biotechnol Res J 2021;5:389-97 |
How to cite this URL:
Shirmohammadi N, Khodaee A, Rahimi M, Vanayi M, Aghanouri R. Formulation of new intelligent nanoparticle inhibited H1N1 influenza subtype and SARS coronavirus type 2 (COVID-19) in vitro. Biomed Biotechnol Res J [serial online] 2021 [cited 2023 Mar 28];5:389-97. Available from: https://www.bmbtrj.org/text.asp?2021/5/4/389/332461 |
| Introduction | |  |
Coronaviruses are a large family of viruses and belong to Coronaviridae which contain cold viruses to more acute viruses such as SARS, MERS, and COVID-19. The latest is the new discovery in man in Wuhan, China, in December 2019. The problem that scientists face while producing the vaccine is mutation of the virus. The SARS-CoV-2 is so intelligent and it can adopt itself with difficult situations easily by changing the genome and mutations are caused by these changes. It is well known that the entry of this intelligent virus is mainly mediated by the interaction between the virus glycoprotein (S1) (a structural protein of the virus) and the angiotensin-converting enzyme 2 (ACE2) in the open state (ACE2 has two open and close states and entry is occurred only in open state). ACE2 is a metallopeptidase enzyme that is attached to the lungs, blood vessels, kidneys, and intestines and it may play a major role in entry of the virus due to stereochemical reasons. Scientists in this work were carried out to solve the problem by concentration on this entry, because sources agree that the viral receptor in human body (ACE2) has similar interactions in all mutations. The main function of drug is that the virus has more affinity to the drug compared to ACE2 (because of magnetic property and iron affinity of virus), so the probability of attachment and entry of virus would be decreased highly. Using nanoparticles decreases the risk of immune responses. On the other hand, the use of magnetic nanoparticles has become increasingly attractive in recent years, because they can be routed to any tissue by magnetic field and therefore they would be more controllable than other nanoparticles.
Metal oxide nanoparticles[1] with their novel properties have had an increasing interest for these kinds of biomedical applications. In recent years, monodispersed superparamagnetic iron oxide nanoparticles have been developed for various biological applications including bioseparation, tissue repair, drug administration, protein purification, biosensing, and MRI and hyperthermia therapy[1] in this work, by functionalizing of particles with polymers,[2],[3] and amino acids the cell toxicity was decreased[4] it is approved by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide [MTT] test), so the drug is bioaffinitive.[5],[6] Furthermore, interfering in viral signaling by magnetism property and quantum-based interactions, can have a big role in inhibitory effect. Briefly, this work suggested a bioaffinitive specific intelligent drug for COVID-19 which can stop the pandemic situation of virus.
| Methods | | |
Preparation of magnetite nanoparticles
For the synthesis of Fe3O4 nanoparticles, coprecipitation method was used as previous methods.
Methods for synthesis of polymer-amino acid-coated superparamagnetic Fe3O4 nanoparticles
- Synthesis of iron oxide nanoparticles modified by a mixture of amino acids
- Synthesis of iron oxide nanoparticles modified by a solution of amino acid– poly(ethylene glycol), PEG
- Synthesis of iron oxide nanoparticles, modification by amino acids, and coating by PEG
- Synthesis of iron oxide nanoparticles, coating by PEG, modification by amino acids.
Synthesis of iron oxide nanoparticles modified by amino acids
In this method, first, 0.03 g of prepared iron oxide nanoparticle was dissolved in 1-ml deionized water by magnetic mixer. 0.4 g of each amino acid was weighted and then dissolved in 1-ml deionized water. To modify nanoparticles by amino acids, the solution which contains a mixture of amino acids was added to previous solution. To separate the precipitation of nanoparticles modified by amino acids, they were centrifuged by refrigerating centrifuge with 12,000 g for 10 min. Then, the precipitation was put in dryer oven for 24 h at 70 c, and finally, the powder of amino acid-modified nanoparticles was produced.
Synthesis of iron oxide nanoparticles modified by PEG–amino acids
First, 0.03 g of iron oxide nanoparticles was dissolved in 1 ml of deionized water by magnetic mixer. Then, 5 mg of PEG was dissolved in 45 ml of deionized water. 0.4 g of each amino acid was dissolved in 1-ml deionized water by magnetic mixer. Then, to make the mixture, two solutions were added to each other and polymer–amino acid mixture was produced. Finally, the mixture was added to Iron oxide nanoparticles and put in centrifuge for 12,000 g for 10 min. Then, obtained mixture was put in freezer at −70 c for 24 min. After that, the mixture was put in freeze dryer for another 24 h. Finally, a powder contains amino acid–PEG-modified nanoparticles.
Synthesis of iron oxide nanoparticles, modification by amino acids and then coating by PEG
In this method, first, 0.03 g of iron oxide nanoparticles was dissolved in 1 ml of deionized water by magnetic mixer. Then, 0.4 g of each amino acid was dissolved in 1-ml deionized water. Then, to modify nanoparticles by amino acids, the amino acid-contained solution was added to prepared solution and it was put in refrigerating centrifuge with 12,000 g for 10 min. The obtained solution was put in 70 c for 24 h until the final powder was produced. 5 mg of PEG polymer was dissolved in 45-ml deionized water by a magnetic mixer. Then, the dried obtained powder from oven which contains amino acid-modified nanoparticles was added to the solution of PEG and was put for 10 min with 12,000 g in refrigerating centrifuge. Finally, obtained solution was again heated in 70 c for 24 h.
Synthesis of iron oxide nanoparticles, coating by PEG, modification by amino acids
In this method, first, 0.3 g iron oxide nanoparticle was dissolved in 1 ml of deionized water by magnetic mixer. To coat nanoparticles by PEG polymer, 5 mg of PEG was dissolved in 45-ml deionized water by magnetic mixer. Then, the PEG polymer solution was added to the solution of nanoparticles and was put in refrigerating centrifuge in 12,000 g for 10 min. The obtained solution was put in oven dryer for 24 h. 0.4 g of each amino acid was dissolved in 1-ml deionized water by magnetic mixer. Then, the obtained powder which contains PEG-coated nanoparticles was added to the solution of amino acid mixture and was put in refrigerating centrifuge with 12,000 g for 10 min. Finally, this solution was again put in oven dryer for 24 h in 70 c to obtain the product.
Cell viability assay
A549 cells (adenocarcinomic human alveolar epithelial cells) were seeded in a 96-well tissue cultural-treated plate with concentration of 104 cells per well. The samples were diluted with DMSO and sonicated for 30 s. The cell viability of the MTT assay was performed with postexposure of 24, 48, and 72 h. The percentage of cell viability was calculated using the following equation. % of proliferation = (Abs620 [treated]/Abs620 [untreated cells]) × 100.
Cytopathic assay
The prepared nanoparticles in four forms were exposed to H1N1 virus for 90 s in temperature of 25°C with concentration of 70 μg/ml analyzes by ISIRI 16676 reference method in Viromed Laboratory of Tehran, Iran, in standard biosafety-level needs. Source of influenza virus was a/Michigan/45/2015/(H1N1) pdm 09-like virus – NBSC code: 16/365.
| Results | | |
To examine and characterize the nanoparticles functionalized by amino acids and coated by poly ethylene glycol, in preclinical step, tests were done as below:
- Chemical properties (Fourier transform infrared [FTIR])
- Morphology and size of particles (field emission scanning electron microscope [FESEM])
- Magnetic properties (vibrating sample magnetometer [VSM])
- Cellular toxicity (MTT)
- Cytopathic test.
Determination of chemical properties by FTIR spectroscopy
The samples were analyzed by ALPHA FTIR spectrophotometer at ARAK University to assure bindings, coating by PEG, and absorption of amino acids by nanoparticles, and results were obtained as below.
Blank sample
One peak was observed in the spectrum of this sample (contains Fe3O4 nanoparticles) at 570.5/cm which shows the existence of iron oxide nanoparticles in the sample [Figure 1].
Sample 1
Main peaks of the spectra related to this sample (obtained by adding L-asparagine, L-serine, L-lysine, and L-glutamic acids to nanoparticles) were observed as below. It was a peak at 593.65/cm (Fe3O4 nanoparticles), 1616.78/cm (amide group of L-asparagine amino acid), and the 3236.06–3550.68 per cm area (amine group of amino acids) [Figure 2].
Sample 2
Main peaks of the spectra related to this sample (obtained by coating and functionalizing by PEG and amino acids simultaneously) were observed as below.
577.91/cm (Fe3O4 nanoparticles), 844.25/cm (C–O bond related to PEG), 949.36/cm (acidic OH), 1637.76/cm (amide group of L-asparagine), 1720.95/cm (C = O bond related to carboxylic acid group), 2864.83/cm (acidic OH), and 3414.08–3460.99 per cm area (amine group of amino acids) [Figure 3].
Sample 3
Main peaks related to the spectra of this sample (obtained by coating the amino acid-functionalized nanoparticles by PEG) were observed as below:
585.42/cm (Fe3O4 nanoparticles), 841.59/cm (C–O bond related to PEG), 1248.48–1281.24 per cm (acidic OH), 1637.94/cm (amide group of L-asparagine amino acid), 2673/cm (wide peak of acidic OH), and 3414.06–354315 per cm area (amine group of amino acids) [Figure 4].
Sample 4
Main peaks of the spectra related to this sample (obtained by functionalizing the PEG-coated Fe3O4 nanoparticles by amino acids were observed as below 568.57/cm (Fe3O4 nanoparticles), 1104.08/cm (acidic OH), 1634.67/cm (amide group of L-asparagine), 1719.07 (C = O bond related to carboxylic acid), 287438/cm (acidic OH), and 3383.52/cm (amine group of amino acids) [Figure 5].
Morphology and particle size analysis by field emission scanning electron microscope
Samples were analyzed by TESCAN MIRA3 FESEM made by Czech Republic owned by Sharif University to get information about morphology and particle sizes. The average particle size was 50 nm. Results were as below.
Blank sample
Magnetite nanoparticles (Fe3O4) without coating and amino acid agent [Figure 6]. | Figure 6: Field emission scanning electron microscope picture of blank sample
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Sample 1
It contains amino acid-functionalized magnetite nanoparticles. The functionalizing of nanoparticles by amino acids can be observed easily [Figure 7]. | Figure 7: Field emission scanning electron microscope picture of Sample 1
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Sample 2
It contains nanoparticles simultaneously coated by PEG and functionalized by amino acids [Figure 8]. | Figure 8: Field emission scanning electron microscope picture of Sample 2
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Sample 3
It contains nanoparticles first functionalized by amino acids and then coated by PEG [Figure 9]. | Figure 9: Field emission scanning electron microscope picture of Sample 3
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Sample 4
It contains nanoparticles first coated by PEG and then functionalized by amino acids [Figure 10]. | Figure 10: Field emission scanning electron microscope picture of Sample 4
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Magnetic properties analyze by vibrating sample magnetometer
Due to magnetic characterization of Fe3O4 nanoparticles and effects of coating by PEG and functionalizing by amino acids on magnetic properties, the properties were analyzed by VSM and results were as below.
Blank sample
It contains Fe3O4 nanoparticles, magnetic saturation was about 90 emu/g, and magnetic hysteresis and coercivity were 0 [Figure 11].
Sample 1
It contains Fe3O4 nanoparticles and L-lysine, L-serine, L-glutamic acid, and L-asparagine; magnetic saturation was about 88 emu/g, and magnetic hysteresis and coercivity were 0 [Figure 12].
Sample 2
It contains nanoparticles simultaneously coated by PEG and functionalized by amino acids, magnetic saturation was about 22 emu/g, and magnetic hysteresis and coercivity were 0 [Figure 13].
Sample 3
It contains nanoparticles first functionalized by amino acids and then coated by PEG, magnetic saturation was about 5 emu/g, and magnetic hysteresis and coercivity were 0 [Figure 14].
Sample 4
It contains nanoparticles first coated by PEG and then functionalized by amino acids, magnetic saturation was about 20 emu/g, and magnetic hysteresis and coercivity were 0 [Figure 15].
Results of vibrating sample magnetometer test
Based on data provided by VSM tests, the blank sample which contains nanoparticles has the highest magnetic saturation (90 emu/g). Sample 1 which is just functionalized by amino acids again has high magnetic saturation, but a bit lower than blank, so the functionalizing does not decrease the magnetic saturation a lot.
In Sample 2 which the coating and functionalizing occur simultaneously, the magnetic saturation is decreased due to coating by PEG (22 emu/g); in Sample 3 which nanoparticles are first functionalized and then coated, the lowest magnetic saturation is occurred (5 emu/g); and finally in Sample 4 which first the coating and then the functionalizing occur, the magnetic saturation is near Sample 2, but in all samples, we do not have any hysteresis and coercivity.
Cell viability
The MTT assay is a colorimetric assay for assessing cell metabolic activity. NAD (P) H-dependent cellular oxidoreductase enzymes may, under defined conditions, reflect the number of viable cells present. These enzymes are capable of reducing the tetrazolium dye MTT to its insoluble formazan, which has a purple color. Other closely related tetrazolium dyes, including XTT, MTS, and the WSTs, are used in conjunction with the intermediate electron acceptor, 1-methoxyphenazine methosulfate. With WST-1, which is cell impermeable, reduction occurs outside the cell via plasma membrane electron transport. However, this traditionally assumed explanation is currently contended as proof has also been found of MTT reduction to formazan in lipophilic cellular structures without apparent involvement of oxidoreductases. Tetrazolium dye assays can also be used to measure cytotoxicity (loss of viable cells) or cytostatic activity (shift from proliferation to quiescence) of potential medicinal agents and toxic materials. MTT assays are usually done in the dark since the MTT reagent is sensitive to light [Figure 16], [Figure 17], [Figure 18], [Figure 19], [Figure 20]. | Figure 16: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide chart dosage 20 μg/ml
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 | Figure 17: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide chart dosage 30 μg/ml
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 | Figure 18: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide chart dosage 50 μg/mls
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 | Figure 19: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide chart dosage 70 μg/ml
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 | Figure 20: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide chart dosage 90 μg/ml
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Cytopathic effect or cytopathogenic effect
It refers to structural changes in host cells that are caused by viral invasion. The infecting virus causes lysis of the host cell or when the cell dies without lysis due to an inability to replicate. Both of these effects occur due to cytopathogenic effects (CPEs). If a virus causes these morphological changes in the host cell, it is said to be cytopathogenic. Common examples of CPE include rounding of the infected cell, fusion with adjacent cells to form syncytia, and the appearance of nuclear or cytoplasmic inclusion bodies [Table 1].
| Discussion | | |
In the recent years, nanomedicine play a new specific role in management of incurable diseases like cancer and viral infections.[7] The novel structure and function of SARS-CoV-2 lead to a lack of validated biomarkers to therapeutic processes.[8] To accomplish this, bio-nanotechnology plays a smart role and could develop next-generation approach to combat the COVID-19 pandemic.[9] Some data demonstrated that the SARS-CoV-2 is inactivated on Cu surfaces and at a temperature higher than 40°C.[10] Furthermore, previous studies showed that the photosensitive TiO2 nanoparticle degrades bacteria and viruses upon photostimulation.[11] By this, new trials were made to design nanoparticles that can affect the SARS-CoV-2. In this work, magnetic nanoparticles were used, due to their simple recognition and direction in human body, simple purification, and magnetic property. It has been suggested that if scientists can investigate a therapeutic agent against the SARS-CoV-2, then it is site-specific delivery that will be a challenge. This site-specific delivery of an optimized therapeutic agent and controlled release and maintenance of the therapeutic agent has a vital role in the possible management of COVID-19.[1],[12] We used magnetic nanoparticles to have a simpler control and management. Regard other studies, we suggested that the SARS-COV-2 can be uptaken by a specific cell because it can securely bind with this virus and cell surface receptors.[13] Our pattern completely follows this target and affects the adhesion of virus to cellular receptor, especially spike proteins to the ACE receptors. Developing an effective therapy for COVID-19 will therefore be a compartmentalization-based approach that will recognize and eradicate the virus and provide support for quick recovery. The synthesized nanoparticles in this work can inhibit the signaling process of virus, and due to affinity of virus to drug, it would prefer attaching to the drug.
Since drug samples were prepared by four procedures, all their properties were considered to choose the best. In contrast to the other studies using a single-drug form in the primary screens, we have used a quantitative method where four compounds were used in the primary screen instead of a single-compound production way. We also assessed the cytotoxicity of each compound against Vero E6 cells (without virus infection) in parallel with the SARS-CoV-2 CPE screening. The concentration-response for each compound used in the primary screen can improve the identification of positive hits. Results of MTT assay approved that Samples 1 and 2 have less cell toxicity, and by cytopathic assay, again, it was shown that Samples 1 and 2 can decrease the virus titer by 0, so totally the decision was taken to use Sample 1 or 2 as the final structure [Table 2]. More detailed results are shown below [Table 2].
Furthermore, because the most concentration of viral receptor is in lungs, so it is logical to prefer formulating the powder as nebulizer spray. On the other hand, high bioavailability of nebulizer, nanoscale structure, and hydrophobicity properties which were measured by Log P factor made us to decide to prefer formulation of nebulizer. We hope that it helps medical society to control the disease.
| Conclusions | | |
The specific intelligent COVID-19 drug was synthesized and analyzed in this work. In order to synthesize, modified Fe3O4 nanoparticles were prepared by coprecipitation method. They were characterized for different properties such as magnetic property, morphology, toxicity, and mainly cytopathic assay. Results show that they had proper magnetic property and they were nontoxic by MTT assay on A549 cell lines. Due to special structure of these nanoparticles that produced in this investigation, inhibit virus entry ( as mentioned in results), Thereby we proposed this kind of drugs can have inhibitory effect on SAR Coronavirus type 2. AS a results, use of these nanoparticles might have high potential for treatment and prevention of COVID-19.
Acknowledgment
We thank Zahra Farahani, Dr. Mohammad Javad Hallaji, and Reza Gheshlaghi for their cooperation in carrying out the project. Danesh Bonyan Garsha Pajooh Scientific-Based Company and Kian Asaye Pars Center for Preventive Medicine and Health Promotion have provided financial support for the project.
Ethical approval
The study was approved by scientific committee in Department of Scientific Research, Kian Asa Center for Preventive Medicine (None Governmental Center Licensed by the Ministry of Health and Medical Education of Iran), SBMU Supervised Area, Tehran, Iran. Scientific Authority Center for Countering Biological Threats, Tehran, Iran. dated 2019.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9], [Figure 10], [Figure 11], [Figure 12], [Figure 13], [Figure 14], [Figure 15], [Figure 16], [Figure 17], [Figure 18], [Figure 19], [Figure 20]
[Table 1], [Table 2]
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