key: cord-0840131-y555mrw1 authors: Jermy, B. Rabindran; Ravinayagam, V.; Almohazey, D.; Alamoudi, W.A.; Dafalla, H.; Akhtar, Sultan; Tanimu, Gazali title: PEGylated green halloysite/spinel ferrite nanocomposites for pH sensitive delivery of dexamethasone: A potential pulmonary drug delivery treatment option for COVID-19 date: 2021-11-09 journal: Appl Clay Sci DOI: 10.1016/j.clay.2021.106333 sha: 3b61c33d4028db1f82a91b5aa97ae76eb74d312d doc_id: 840131 cord_uid: y555mrw1 Dexamethasone (Dex) is used in drug regimen for treatment of Coronavirus disease (COVID-19). Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) fusion and entry into the cell occurs at pH 5.5. In our present study, we have identified a green, cheap clay based halloysite (Hal) nanoformulation with release capability of Dex at such interactive pH condition. 30%ZnFe(2)O(4)/Hal and 30%NiFe(2)O(4)/Hal were prepared by one-pot synthesis technique. Dex (5% wt/wt) was functionalized over both nanocomposites. Finally, polyethylene glycol (PEG) was coated over ZnFe(2)O(4)/Hal/Dex and NiFe(2)O(4)/Hal/Dex nanocomposite using lyophilization technique (0.08 μl/mg of nanocarrier). The release ability of Dex was studied under pulmonary infection and normal pH conditions (pH = 5.6 and 7.4). The characterization study using X-ray diffraction (XRD) and UV–visible diffuse reflectance (DRS) spectra confirmed the presence of spinel ferrites over Hal. Nitrogen adsorption isotherm showed the surface area of ZnFe(2)O(4)/Hal (75 m(2)/g), pore volume (0.27 cm(3)/g) with average pore size (14.5 nm). Scanning electron microscope/Energy dispersive spectroscopy (SEM-EDS) and Transmission electron microscopy analysis revealed a textural change in halloysite tubular type indicating drug adsorption and PEG adhesion. DRS spectra indicated an intergrowth of zinc ferrite nanoparticles on the halloysite nanotubes. Interestingly, ZnFe(2)O(4)/Hal/Dex/PEG exhibited a high Dex release ability (17.5%, 168 h) at pH = 5.6 relevant to SARS-CoV-2 fusion entry into the cell pH condition of 5.5. Comparatively, the nanocomposite showed a less Dex release (<5%) release for 168 h at neutral pH = 7.4. The drug release kinetics were studied and the obtained data were fitted for the release constant and release exponent, using the Korsmeyer-Peppas model. To test the compatibility of our nanocomposites, we performed the cell viability assay (MTT) using HEK293 cells. Our results showed that at 0.3 mg/ml, Dex-loaded nanocomposite had a statistically significant improvement in cell viability compared to Dex alone. These results suggest that our nanocomposite has prevented the toxic effect of Dex and has huge potential to act as pulmonary drug delivery system for targeted lung infection therapeutics. Coronavirus belonging to large family of viruses cause illnesses of a wide range of severity. COVID-19 is caused by the severe acute respiratory syndrome corona virus 2 (SARS-CoV-2). COVID-19 pandemic related mortality is continuously increasing irrespective of the preventive measures taken worldwide. Up to now, 187. 8 The treatment strategies of SARS-CoV-2 infection includes drugs that specifically target host cell interactions or proteins generated by virus [3] . The proteins evolution of virus effectively targets the receptor termed as angiotensin-converting enzyme (ACE2), a prime target identified for nanotherapeutic intervention (Bonam et al., 2021) . Up to now, there is no effective antiviral drug available to combat COVID-19. One strategy involves blocking the virus entry by blocking specific CoV receptors using protease inhibitors (Camostat mesylate, Umifenovir, lopinavir/ritonavir) ( However, the treatment with dexamethasone also impart several side effects like hormonal J o u r n a l P r e -p r o o f Journal Pre-proof disturbance, oedema, weight gain, sleep disorder and dose related adverse side effects (Cole, 2020) . Dexamethasone also cause diabetes related complications, immune suppressor and cause hypertension. Poor gastrointestinal stability, low bioavailability, poor transport behavior and side effect on other organs like kidney limit its therapeutic effectivity (Alessi et al., 2020; Huang et al., 2017) . Pulmonary drug delivery research has been rising steadily in interdisciplinary core area for treatment of lung diseases. Such therapeutic route increases the targeted drug delivery, reduces the effective dose, easily adsorbed on available large lung surface area (Rangaraj et al., 2019; Strzempek et al., 2019) . The most striking features of pulmonary nanotherapeutics is the ability of nanoparticle to accommodate several components into nano structure to generate multifunctional modality. Pulmonary drug delivery system has been developed to treat of lung infection asthma, chronic pulmonary diseases. One of the prerequisite of such delivery system is that it should able to increase the bioavailability, strong adhesion ability, rapid diffusion through mucus, sustained drug release capacity and delivering drugs specifically to bronchi and alveoli (Bonam et al., 2021) . For instance, several nanocarriers based on metal organic framework Fe-MIL-100 (Strzempek et al., 2019) , liposomes (Yıldız-Peköz and Ehrhardt, 2020), micelles (Pham et al., 2021) , and inorganic nanoparticles (Xu et al., 2020) has been reported for treating pulmonary infections. Magnetically active micro or nanoparticles, superparamagnetic iron oxide nanoparticles (SPIONs) based drug delivery system has shown to improve the targeted delivery to desired position with the help of external magnetic field (Saadat et al.,2020; Stocke et al., 2015) . Magnetic microrods fabricated with poly (ethylene oxide) and poly (l-lactide) polymers are shown to increase the drug accumulation and improve targeted release of drugs at the lower airways (Nikolaou et al., 2021) . ACE-2 surface rich membrane based nanomaterials synthesized by extrusion technique was reported to be effective in blocking virus entry into host renal tubular cells and reduce the SARS-CoV-2 infection . Inspite of recent advancement in vaccines and treatment related to antiviral therapy, a continuous rise in the mortality related to COVID-19 clearly shows the need for developing an effective targeted pulmonary drug delivery system. J o u r n a l P r e -p r o o f Journal Pre-proof A nanotechnology tool with diagnostic tool sensing SARS-CoV-2 specific cell entry through interactive pH is yet to be reported. In addition, an effective pulmonary delivery capable of carrying and releasing antiviral agent is required. Dex is used in drug regimen for treatment of COVID-19. High dose increases mortality rate, poor gastrointestinal stability, low bioavailability, poor transport behavior limit its therapeutic effectivity. For example, a low dose administration of dexamethasone is proposed to overcome acute respiratory distress syndrome (Mahase, 2021 Gold nanoclusters supported on silane modified Hal has been described as an effective nanocomposite inducing non-toxic effect at desired concentration range (25-50 µg/ml) suitable for tumor imaging and cancer therapy (Gorbachevskii et al. 2021 ). The present study put forward an important stratergy by designing a nanosystem that can indentify such virus-ACE2 interactions pH 5.5 with a potential to attack the virus by delivering a known antiviral drugs in slow and controlled manner. Zinc ferrite or nickel ferrite/Hal wih magnetic resonance imaging property was investigated for potential pulmonary drug delivery including COVID-19. The textural and chemical environment of both ferrite system and Dex was analyzed using different characterization techniques. The technique is simple, reproducible, and scalable. The study shows that the developed pulmonary drug delivery system is more effective in releasing drug Dex at pulmonary infectious pH condition of 5.6 matching with the pH of virus interacting with ACE-2 receptor. J o u r n a l P r e -p r o o f Then the solution mixture was freeze dried using lyophilization technique. The phase of support carriers such as ZnFe The drug release trend of Dex To assess the compatibility and effectiveness of our nanocomposites, we used the human embryonic kidney cells HEK293 cell lines. Cells were cultured in a DMEM medium with 10 % HI-FBS, 1 % Penicillin Streptomycin, and 1 % MEM NEAA. Cell cultures were maintained in a cell incubator at 37˚C with 5 % CO 2 in humidified conditions. For the MTT cell viability assay, cells were seeded in a 96-well plate with a density of 20,000 cells/well. After 24 h from seeding, cells were treated with the conditions and treatment durtation discussed in the following section. We first performed a dose response curve. HEK293 cells were treated with the the We used the MTT cell viability assay to assess the compatibility and effectiveness of our nanocomposites. The assay assesses the viability of cells via evaluation of mitochondrial function as MTT is converted to formazan (Mosmann, 1983) . After washing cells with PBS, MTT working solution was added at a final concentration of 0.5 mg/ml. Cells were incubated at 37˚C for 3 h after which 0.04 N HCl isopropyl alcohol was added to solubilize the formazan crystals. Change in color was measured using SYNERGY-neo2 BioTek ELISA reader at 570 nm. An initial reading (before addition of MTT) was recorded and subtracted from the final reading to avoid background interference from NPs. Each condition was performed in three technical repeats with three biological repeats (n= 3). A no treatment negative control and an MTT background control (no cells) were also included. Analysis was performed by comparing treatment conditions with the no treatment negative control. Percentage of cell viability was measured as follows: Cell Viability (%) = (abrobance of condition / absorbance of negative control) x 100. Cells were plated on a chamber slide and treated with the treatment conditions for 24, 48, and 72 h. Nikon Eclipse TS100 (inverted microscope) was used to capture brightfield images. The Table 1 , the surface area of halloysite was observed to be 76 m 2 /g (Fig. 2a) , which only slightly changes to 75 m 2 /g after nanocomposite formation with zinc ferrite. However, the pore volume decreases from 0.33 cm 3 /g to 0.27 cm 3 /g and average pore size distribution from 17.7 nm to 14.5 nm, respectively (Fig. 2b) . J o u r n a l P r e -p r o o f (Fig. 5a) . In case of freeze dried ZnFe 2 O 4 /Hal/Dex/PEG sample, addition of Dex and PEG clearly changed the textures of tubular clay indicating the drug embendment and PEG adhesion (Fig. 5b) . 5g and h). The tubalr nature of the clay is judged by a brigh contrast of each clay particle as compared to their outer dark sides. The size of the nanoparticles is measured by extracting the intensity profile of the indicated particle as marked with a yellow arrow (Fig. 5i) Fig. 5j and k) . Furthermore, the tubular morphology (bright contrast) of the clay/nanoparticles is less visible due to coating of PEG. SAED pattern confirmed the coexistance of spinel ferrite nanoparticles with clay, the possible diffracting reflections of Dex/PEG over ZnFe 2 O 4 /Hal nanoclay are indexed as: (220), (311), (422) and (440) (see Fig. 5l ). In summary, TEM analysis confirming the successful preparation of ZnFe 2 O 4 decorated tubular clay and coating of Dex/PEG over this composite. Where R% is the drug percentage release at time (t), k and n are the kinetic rate constant and the release exponent, respectively. The obtained fitting parameters together with their 95% confidence intervals are shown in Table 2 . reduced with decrease in the pH value. This indicates that, the rate of drug release using this material is enhanced with increase in pH. The release exponent (n) signified fickian (n < 0.45) and non-fickian (0.45 < n < 0.89) diffusion mechanisms for pH= 7.4 and 5.6, respectively. Increase in pH reduces the rate of drug release using NiFe 2 O 4 /Hal. The pH has no effect on the diffusion mechanism as both release exponents n < 0.45, confirming fickian mechanism. For ZnFe 2 O 4 /Hal/PEG, lower pH increases the rate of drug release as observed from the higher rate constant. The release exponent (n) at both pH signifies fickian diffusion mechanism. Interestingly, for NiFe 2 O 4 /Hal/PEG, the kinetic rate constant increases with increase in pH, signifying improved rate of drug release at higher pH. Fickian diffusion mechanism was followed at all the two pH. Conclusively, adding PEG to the composite materials increases the rate of drug release especially for ZnFe 2 O 4 /Hal material as observed from the kinetic rate constants of the before and after adding PEG. J o u r n a l P r e -p r o o f (Fig. 7) . Then we selected specific concentratios to be treated at different time points. Cells were treated with dose 2 (0.075 mg/ml- Fig. 8 ), or dose 4 (0.3 mg/ml- Fig. 9 ) for 24, 48, and 72 h. Upon analysis of our results, we decided that dose 4 (0.3 mg/ml) was the optimal dose as it shows a distinct separation between the cell viability curves. This was confirmed with statistical analysis of dose 4 treatment for 24, 48, and 72 h ( Fig. 9 .B,C,D) and the morphological assessment of treated cells (Fig. 10 COVID-19 is caused by the severe acute respiratory syndrome corona virus 2 (SARS-CoV-2). One of treatment strategy is developing a pulmonary drug delivery system with capability of delivering antiviral drugs specifically targeting host cell interactions or proteins generated by virus. The nanocarrier particles with sizes between 60-300 nm is reported to diffuse freely in tracheal mucus of mouse. Biocompatible polymer polyethylene glycol coating of nanoparticle of 100 and 200 nm shown to penetrate respiratory mucus. In present study, a green and eco-friendly clay based halloysite with particle size 50-70 nm have been successfully used as pulmonary nanocarrier for delivery of Dex. ZnFe 2 O 4 /Hal and NiFe 2 O 4 /Hal nanocomposites are successfully synthesized using one-pot method. The technique is simple, reproducible, and scalable. X-ray diffraction, diffuse reflectance and elemental mapping analysis showed the cubic spinel structure with particle size ranging between 6-8.6 nm. FTIR confirmed the vibrations of spinel ferrite, Dex conjugation and PEGylation over halloysite. The study shows that the developed ZnFe 2 O 4 /Hal/DEX/PEG drug delivery system is more effective in releasing drug Dex at pulmonary infectious pH condition. It is known that Coronovirus entry into the cell is a pH dependent process (ACE-2 receptor mediated process). SARS-Cov-2 fusion, entry into the cell occurs at pH 5.5. Hence our drug carrier which is sensitive to pH 5.6, could effectively delivery drug at this point of pH dependent process. The drug release kinetics at the studied pH values revealed that, the rate of drug release is dependent on the spinel ferrite/Hal nanocomposite and diffusion mechanism is mostly independent of the pH. Our MTT results showed that Dex-loaded nanocomposite at 0.3 mg/ml had a statistically significant improvement in cell viability and prevented the toxic effect of Dex. Therefore, the system can be used as pulmonary drug delivery of nanomedicine like dexamethansone, and other potential antiviral drugs. 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Rabindran Jermy developed the idea, formulated, characterized spinel ferrite/Hal/PEG nanocomposite, characterizations, acquire financial support. Vijaya Ravinayagam developed the conceptualization of drug formulation, delivery, in vitro, interpretation. Dana Almohazey, in vitro study, dose optimization, experimental discussion and writing part. Ms. Alamoudi, involved in in vitro experimental part. H. Dafalla performed the SEM-EDX, elemental mapping analysis. Dr. Sultan Akhtar performed TEM experiments, analysis and wrote discussion part relevant to microscopic technique. Gazali Tanimu performed kinetics study for drug release. All authors reviewed and approved the final manuscript. ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:J o u r n a l P r e -p r o o f