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. 2021 Jun:34:102286.
doi: 10.1016/j.pdpdt.2021.102286. Epub 2021 Apr 7.

Robust antimicrobial photodynamic therapy with curcumin-poly (lactic-co-glycolic acid) nanoparticles against COVID-19: A preliminary in vitro study in Vero cell line as a model

Maryam Pourhajibagher  1 , Maryam Azimi  2 , Vahid Haddadi-Asl  3 , Hanie Ahmadi  3 , Mehrdad Gholamzad  4 , Sara Ghorbanpour  5 , Abbas Bahador  6
Affiliations

Robust antimicrobial photodynamic therapy with curcumin-poly (lactic-co-glycolic acid) nanoparticles against COVID-19: A preliminary in vitro study in Vero cell line as a model

Maryam Pourhajibagher et al. Photodiagnosis Photodyn Ther. 2021 Jun.

Abstract

Background: In this study, the ability of antimicrobial photodynamic therapy (aPDT) as a treatment approach and adjuvant therapy using curcumin-poly (lactic-co-glycolic acid) nanoparticles (Cur@PLGA-NPs) to inactivate Coronavirus disease 2019 (COVID-19) in plasma was investigated. Furthermore, to verify whether the quality requirement of aPDT-treated plasma is acceptable, the differences of the levels of clotting factors, total plasma proteins, and anti-A and/or anti-B antibodies titrations in plasma of patient before and after aPDT treatment were investigated.

Materials and methods: Cur@PLGA-NPs was synthesized using Electrospinning process and characterized by different analysis including Scanning Electron Microscope (SEM), Transmission Electron Microscope (TEM), and Fourier Transform Infrared (FTIR) spectroscopy assays. The presence of the SARS-CoV-2 in the plasma samples of patients suspected of having COVID-19 was confirmed by real-time reverse transcription-polymerase chain reaction (RT-PCR) assay. Then, the treated plasma samples with Cur@PLGA-NPs plus blue laser were exposed to Vero cells. Eventually, cell cytotoxicity and apoptotic effects of treated Vero cells were evaluated. Levels of clotting factors including prothrombin time (PT) and activated partial thromboplastin time (APTT), total plasma proteins, and anti-A and/or anti-B antibodies measurements were performed using the coagulometer, method of Bradford, and titration procedure, respectively.

Results: The presence of SARS-CoV-2 was positive in 84.3 % of samples. Different concentrations of Cur@PLGA-NPs (3, 5, 7, and 10 % wt.), the irradiation times of blue laser (1, 3, and 5 min), and aPDT with the maximum dosed of blue laser light (522.8 J/cm2) plus 10 % wt. Cur@PLGA-NPs had no cytotoxicity. Although there were significant cell degradation and apoptotic effects in treated Vero cells with treated plasma using 10 % wt. Cur@PLGA-NPs, and a blue laser at an energy density of 522.8 J/cm2, no visible changes in cells and apoptosis were observed following aPDT. Total plasma protein content, PT, APTT, and anti-A and/or anti-B antibodies titers showed no significant changes (P > 0.05 for all comparisons) in treated plasma as compared to untreated plasma.

Conclusion: aPDT exhibited in vitro anti-COVID-19 activities in the treated plasma containing SARS-COV-2 without Vero cell apoptosis and any adverse effects on plasma quality in aPDT-exposed plasma.

Keywords: Antimicrobial photodynamic therapy; COVID-19; Coronavirus; Curcumin; PLGA; SARS-CoV-2.

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Conflict of interest statement

The authors declare that there is no conflict of interests regarding the publication of this paper.

Figures

Fig. 1
Fig. 1
The schematic diagram of Cur@PLGA-NLs synthesis.
Fig. 2
Fig. 2
The blue laser device used in this study.
Fig. 3
Fig. 3
Semi-auto coagulation analyzer (coagulometer) used to test the coagulation factors (PT and APTT).
Fig. 4
Fig. 4
Anti-A and/or anti-B antibodies measurements were performed using the titration procedure where the RBC remained agglutinated after gentle shaking.
Fig. 5
Fig. 5
Characterization of the synthesized Cur@PLGA-NLs; a) SEM image of Cur@PLGA-NLs (scale bar represents 1 μm), b) TEM image of Cur@PLGA-NLs at different magnification, Left image with less magnification (scale bar represents 250 nm), right with more magnification that shows the core-shell structure well (scale bar represents 20 nm), c) FT-IR spectra of PLGA (pink), Cur (yellow), Cur@PLGA-NLs (blue).
Fig. 6
Fig. 6
UV–vis absorption spectra of Cur@PLGA-NLs.
Fig. 7
Fig. 7
Measurement of physical stability of Cur@PLGA-NPs during storage using particle size, polydispersity index, and zeta potential of Cur@PLGA-NLs in different conditions; a) at 4 °C, b) at 37 °C.
Fig. 8
Fig. 8
Confirmation of the presence of SARS-CoV-2 by detection of ORF1ab gene: A) The cDNA template synthesized from ORF1ab on the 2% gel agarose: 1. Ladder 50 bp, 2. Control positive (108 bp), 3 and 4. Two positive samples (108 bp). B) The amplification curves of positive samples: 1. Control positive, 2 and 3. Two positive samples.
Fig. 9
Fig. 9
Determination of the Vero cells viability by MTT assay at 540 nm; I) Treated Vero cells with different concentrations of Cur@PLGA-NLs, II) Treated Vero cells with different irradiation times of blue laser light, and III) Treated Vero cells with aPDT using 10 % wt. Cur@PLGA-NPs plus blue laser light at an energy density of 522.8 J/cm2.
Fig. 10
Fig. 10
Determination of the cell cytotoxicity of Vero cells with treated plasma containing SARS-CoV-2 by MTT assay at 540 nm; P: Untreated plasma containing SARS-CoV-2, P*: Treated plasma containing SARS-CoV-2, PS: Photosensitizer (10 % wt. Cur@PLGA-NPs), aPDT: 10 % wt. Cur@PLGA-NPs plus blue laser light at an energy density of 522.8 J/cm2. Significant differences according to the control. * P < 0.05.
Fig. 11
Fig. 11
Determination of the apoptotic effects in treated Vero cells with treated plasma containing SARS-CoV-2; A) The apoptotic ratio was assessed by flow cytometry with Annexin V staining in Vero cells: a. Untreated Vero cells (as a control group), b. Treated Vero cells with untreated plasma containing SARS-CoV-2, c) Treated Vero cells with treated plasma containing SARS-CoV-2 using 10 % wt. Cur@PLGA-NPs, d) Treated Vero cells with treated plasma containing SARS-CoV-2 using blue laser light at an energy density of 522.8 J/cm2, e) Treated Vero cells with treated plasma containing SARS-CoV-2 using aPDT with 10 % wt. Cur@PLGA-NPs plus blue laser light at an energy density of 522.8 J/cm2. B) Percent of cell viability at 540 nm. P: Untreated plasma containing SARS-CoV-2, P*: Treated plasma containing SARS-CoV-2. PS: Photosensitizer (10 % wt. Cur@PLGA-NPs). Significant differences according to the control, * P < 0.05.
Fig. 12
Fig. 12
Determination of the apoptotic effects in exposed Vero cells by acridine orange /ethidium bromide fluorescent staining under the fluorescent microscope (OLYMPUS BX53, Japan; objective magnification 10x; Scale bars=100 μm): a) Untreated Vero cells, as a control group, revealing green nuclear staining, b) Exposed Vero cell culture with untreated plasma containing SARS-CoV-2 showing a large number of apoptotic cells which had orange and red nuclear fluorescence emission, c) Exposed Vero cell culture with treated plasma containing SARS-CoV-2 using 10 % wt. Cur@PLGA-NPs, indicating decrease in cells with orange-red nuclear staining in comparison to exposed Vero cell culture with untreated plasma containing SARS-CoV-2, d) Exposed Vero cell culture with treated plasma containing SARS-CoV-2 using blue laser light (522.8 J/cm2) representing increase the number of apoptotic cells more than exposed Vero cells with treated plasma containing SARS-CoV-2 using 10 % wt. Cur@PLGA-NPs and less than exposed Vero cells with untreated plasma containing SARS-CoV-2, e) Exposed Vero cells with treated plasma containing SARS-CoV-2 using aPDT with 10 % wt. Cur@PLGA-NPs plus blue laser light (522.8 J/cm2) demonstrating a significant reduction in the number of apoptotic cells in comparison to exposed Vero cells with untreated plasma containing SARS-CoV-2.
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