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. 2020 Jul 22:15:5181-5202.
doi: 10.2147/IJN.S247692. eCollection 2020.

Virucidal Action Against Avian Influenza H5N1 Virus and Immunomodulatory Effects of Nanoformulations Consisting of Mesoporous Silica Nanoparticles Loaded with Natural Prodrugs

Affiliations

Virucidal Action Against Avian Influenza H5N1 Virus and Immunomodulatory Effects of Nanoformulations Consisting of Mesoporous Silica Nanoparticles Loaded with Natural Prodrugs

Khaled AbouAitah et al. Int J Nanomedicine. .

Abstract

Background: Combating infectious diseases caused by influenza virus is a major challenge due to its resistance to available drugs and vaccines, side effects, and cost of treatment. Nanomedicines are being developed to allow targeted delivery of drugs to attack specific cells or viruses.

Materials and methods: In this study, mesoporous silica nanoparticles (MSNs) functionalized with amino groups and loaded with natural prodrugs of shikimic acid (SH), quercetin (QR) or both were explored as a novel antiviral nanoformulations targeting the highly pathogenic avian influenza H5N1 virus. Also, the immunomodulatory effects were investigated in vitro tests and anti-inflammatory activity was determined in vivo using the acute carrageenan-induced paw edema rat model.

Results: Prodrugs alone or the MSNs displayed weaker antiviral effects as evidenced by virus titers and plaque formation compared to nanoformulations. The MSNs-NH2-SH and MSNs-NH2-SH-QR2 nanoformulations displayed a strong virucidal by inactivating the H5N1 virus. They induced also strong immunomodulatory effects: they inhibited cytokines (TNF-α, IL-1β) and nitric oxide production by approximately 50% for MSNs-NH2-SH-QR2 (containing both SH and QR). Remarkable anti-inflammatory effects were observed during in vivo tests in an acute carrageenan-induced rat model.

Conclusion: Our preliminary findings show the potential of nanotechnology for the application of natural prodrug substances to produce a novel safe, effective, and affordable antiviral drug.

Keywords: immunomodulatory and anti-inflammatory; influenza H5N1 virus; mesoporous silica nanoparticles; nanoformulations-drug delivery system; shikimic acid and quercetin natural prodrugs; virucidal action.

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Conflict of interest statement

The authors report no conflicts of interest in this work.

Figures

Figure 1
Figure 1
Morphological structure, size measurements, and schematic representation of preparation steps. Notes: TEM image of nanoparticles (A), SEM image of nanoparticles (B), NTA analysis of particle size (C, D, E, F, G), and schematic representation of the synthesis method for every stage (H).First, the nanoparticles were synthesized, then modified with amino groups (-NH2) via a post-synthesis method. Next, prodrugs were loaded onto modified nanoparticles to obtain nanoformulations. For combined nanoformulations, we used MSNs-NH2-SH as the starting material to load quercetin, and the resulting nanoformulation was MSNs-NH2-SH-QR1. Abbreviations: TEM, transmission electron microscopy; SEM, scanning electron microscopy; MSNs, mesoporous silica nanoparticles; MSNs-NH2, MSNs modified amino groups; MSNs-NH2-SH, MSNs-NH2 loaded SH; MSNs-NH2-QR, MSNs-NH2 loaded QR; MSNs-NH2-SH-QR1, MSNs-NH2 loaded SH and QR; SH, shikimic acid; QR, quercetin.
Figure 2
Figure 2
XRD and DSC measurements. Notes: XRD patterns of all prepared materials. Starting from the bottom: nanoparticles, modified nanoparticles, nanoformulations, combined nanoformulations, and pure prodrugs (A).DSC spectra obtained for materials. Starting from the bottom: nanoparticles, modified nanoparticles, nanoformulations, combined nanoformulations, and pure prodrugs (B). Abbreviations: XRD, X-ray powder diffraction; DSC, differential scanning calorimetry analysis; P.M. 2–3D – SH-QR is a physical mixture of prodrugs and MSNs; MSNs, mesoporous silica nanoparticles; MSNs-NH2, MSNs modified amino groups; MSNs-NH2-SH, MSNs-NH2 loaded SH; MSNs-NH2-QR, MSNs-NH2 loaded QR; MSNs-NH2-SH-QR, MSNs-NH2 loaded SH and QR; SH, shikimic acid; QR, quercetin.
Figure 3
Figure 3
STA, FTIR, and zeta potential measurements. Notes: STA analysis of nanoparticles, modified nanoparticles, nanoformulations, and combined nanoformulations (A). The thermal analysis of the dried powders was employed to detect the weight loss from materials (nanoparticles and prodrugs) at high-temperature condition. The dried powder of each sample was heated up to 800ºC leading to lose water content, organic compounds used in surface modification and loaded prodrugs. The MSNs are inorganic material which is stable at high temperature- this help to calculate the drug loading amount. The drug loading % in nanoformulations was calculated based on the weight loss between modified nanoparticles and after loading into nanoformulations. FTIR spectra of nanoparticles, modified nanoparticles, nanoformulations, combined nanoformulations, and pure prodrugs (B). Zeta potential measurements of materials in aqueous suspensions from acidic to alkaline (C). Abbreviations: STA, simultaneous thermal analysis; FTIR, Fourier transform infrared spectroscopy; MSNs, mesoporous silica nanoparticles; MSNs-NH2, MSNs modified amino groups; MSNs-NH2-SH, MSNs-NH2 loaded SH; MSNs-NH2-QR, MSNs-NH2 loaded QR; MSNs-NH2-SH-QR, MSNs-NH2 loaded SH and QR.
Figure 4
Figure 4
Cytotoxicity of materials tested for cell viability by MTT assay in MDCK cells. Notes: As a function of nanoparticle and modified nanoparticle concentrations (A). As a function of prodrug and nanoformulation concentrations equal to pure prodrugs used alone (B). SH/QR mix2 (pure prodrug mixture) and MSNs-NH2-SH-QR2 are the same materials as SH/QR mix1 and MSNs-NH2-SH-QR1 but twice the amount – see Table 1. Antiviral activity against H5N1 virus (C). The safe concentration (equivalent to 75 µg/mL of free compound or found in nanoformulations) was used for all materials. The safe concentration used for unmodified and modified nanoparticles was 50 µg/mL. Data are presented as mean ± SD. *Means significant differences at p <0.05. The concentration for MSNs and MSNs-NH2 was directly made of prepared powder of both nanoparticles. The concentration used in nanoformulations of MSNs-NH2-SH, MSNs-NH2-QR, MSNs-NH2-SH-QR, and MSNs-NH2-SH-QR2 was calculated based on the prodrug loaded into nanoparticles as an equivalent to free prodrugs (SH, QR, SH/QR mix1, and SH/QR mix). The free prodrug concentration was normally made from the powder of pure prodrugs without any calculation. Abbreviations: MTT, cell proliferation assay; MDCK, Madin-Darby Canine Kidney cells; MSNs, mesoporous silica nanoparticles; MSNs-NH2, MSNs modified amino groups; MSNs-NH2-SH, MSNs-NH2 loaded SH; MSNs-NH2-QR, MSNs-NH2 loaded QR; MSNs-NH2-SH-QR1, MSNs-NH2 loaded SH and QR; MSNs-NH2-SH-QR2 was used in twice amount; SH/QR mix.1, mixture of SH and QR; SH/QR mix.2, mixture of SH and QR used in twice amount; SH pure, shikimic acid; QR pure, quercetin; SD, standard deviation.
Figure 5
Figure 5
Plaque reduction assay used to evaluate antiviral activity against H5N1 virus using MDCK cells. Notes: Nanoparticles, modified nanoparticles, prodrugs and their mixtures, nanoformulations, and combined nanoformulations with different post-time infection (A). The safe concentration (equivalent to 75 µg/mL of free compound or found in nanoformulations) was used for all materials. The safe concentration used for unmodified and modified nanoparticles was 50 µg/mL.IC50 for successful nanoformulations (B). a) MSNs-NH2-SH at 25, 50, and 75 µg/mL. b) MSNs-NH2-SH-QR2 at 50, 75, and 150 µg/mL. Data are presented as mean ± SD. * Means significant differences at p <0.05. The concentration for MSNs and MSNs-NH2 was directly made of prepared powder of both nanoparticles. The concentration used in nanoformulations of MSNs-NH2-SH, MSNs-NH2-QR, MSNs-NH2-SH-QR, and MSNs-NH2-SH-QR2 was calculated based on the prodrug loaded into nanoparticles as an equivalent to free prodrugs (SH, QR, SH/QR mix1, and SH/QR mix). The free prodrug concentration was normally made from the powder of pure prodrugs without any calculation. Abbreviations: MSNs, mesoporous silica nanoparticles; MSNs-NH2, MSNs modified amino groups; MSNs-NH2-SH, MSNs-NH2 loaded SH; MSNs-NH2-QR, MSNs-NH2 loaded QR; MSNs-NH2-SH-QR1, MSNs-NH2 loaded SH and QR; MSNs-NH2-SH-QR2 was used in twice amount; SH/QR mix.1, mixture of SH and QR; SH/QR mix.2, mixture of SH and QR used in twice amount; SH pure, shikimic acid; QR pure, quercetin; IC50, the half-maximal inhibitory concentration; SD, standard deviation.
Figure 6
Figure 6
Antiviral mechanisms of nanoformulations tested by plaque reduction assay. Notes: Plaque formation (white dots) appeared or disappeared in response to the tested mode of action of the nanoformulations (A). Left: virus control. Right: treated samples. Schematic representation of the antiviral mechanisms for effective nanoformulations (MSNs-NH2-SHand MSNs-NH2-SH-QR2) developed with mesoporous silica nanoparticles (B). a) Virucidal mechanism. b) Viral replication mechanism. c) Viral adsorption mechanism. Abbreviations: H5N1, high pathogenic avian influenza virus; MSNs, mesoporous silica nanoparticles; MSNs-NH2-SH, MSNs-NH2 loaded SH; MSNs-NH2-QR, MSNs-NH2 loaded QR; MSNs-NH2-SH-QR2, MSNs-NH2 loaded SH and QR and was used in twice amount; MDCK, Madin-Darby Canine Kidney cells.
Figure 7
Figure 7
In vitro cytotoxicity evaluation immune cells and in vivo anti-inflammatory. Lymphocyte cell viability with nanoparticles, modified nanoparticles, prodrugs and their mixtures, nanoformulations, and combined nanoformulations (A). Lymphocyte index for nanoparticles, modified nanoparticles, prodrugs and their mixtures, nanoformulations, and combined nanoformulations (B). Anti-inflammatory activity in carrageenan-induced rats as the change in paw thickness (mm) (C). Data are expressed as mean ± SD. *p<0.05, medium significance (**) and high significance (***). Abbreviations: MSNs, mesoporous silica nanoparticles; MSNs-NH2, MSNs modified amino groups; MSNs-NH2-SH, MSNs-NH2 loaded SH; MSNs-NH2-QR, MSNs-NH2 loaded QR; MSNs-NH2-SH-QR1, MSNs-NH2 loaded SH and QR; MSNs-NH2-SH-QR2 was used in twice amount; SH/QR mix.1, mixture of SH and QR; SH/QR mix.2, mixture of SH and QR used in twice amount; SH pure, shikimic acid; QR pure, quercetin; IC50, the half-maximal inhibitory concentration; SD, standard deviation.

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