Combining molecular dynamics simulations and x-ray scattering techniques for the accurate treatment of protonation degree and packing of ionizable lipids in monolayers

The pH-dependent change in protonation of ionizable lipids is crucial for the success of lipid-based nanoparticles as mRNA delivery systems. Despite their widespread application in vaccines, the structural changes upon acidification are not well understood. Molecular dynamics simulations support structure prediction but require an a priori knowledge of the lipid packing and protonation degree. The presetting of the protonation degree is a challenging task in the case of ionizable lipids since it depends on pH and on the local lipid environment and often lacks experimental validation. Here, we introduce a methodology of combining all-atom molecular dynamics simulations with experimental total-reflection x-ray fluorescence and scattering measurements for the ionizable lipid Dlin-MC3-DMA (MC3) in POPC monolayers. This joint approach allows us to simultaneously determine the lipid packing and the protonation degree of MC3. The consistent parameterization is expected to be useful for further predictive modeling of the action of MC3-based lipid nanoparticles

Synthesis of photoactivable Pt(IV) prodrug loaded on NaYF4 based upconversion nanoparticles functionalized with 2-deoxy-D-glucose and its evaluation for targeted cancer therapy

21-30Nano-formulation based on Tm, Yb doped NaYF4 upconversion nanoparticles (UCNPs) functionalized with 2-deoxy-D-glucose have been synthesized to load the photoactivable Pt(IV) prodrug, cis-[PtI2(NH3)2(OCOCH2CH2COOH)2]. The Pt(IV) prodrug has been synthesized by oxidation of cis-[PtI2(NH3)2] to [PtI2(OH)2(NH3)2] and its further treatment with succinic anhydride. It is loaded through ester bond formation between the carboxyl groups of Pt(IV) prodrug with hydroxyl groups of 2-deoxy-D-glucose (2-DG) coated on UCNPs. The cytotoxicity of formulation after exposing to 385 nm UV light and in absence of light is evaluated against MCF-7 cell lines by MTT assay. The results have revealed enhanced cytotoxicity of UV exposed nano-formulation. Additionally, the clonogenic assay has exhibited the decrease in plating efficiency as inferred from decreased surviving fraction around 20% only for UV activated formulation as compared to formulation in dark, as well as merely Pt(IV) prodrug. These results are indicative that more internalization of the formulation inside the cancer cells was achieved due to the presence of 2-DG rendering more efficiency to kill cancer cells

pH-dependent structural transitions in cationic ionizable lipid mesophases are critical for lipid nanoparticle function

Lipid nanoparticles (LNPs) are advanced core-shell particles for messenger RNA (mRNA) based therapies that are made of polyethylene glycol (PEG) lipid, distearoylphosphatidylcholine (DSPC), cationic ionizable lipid (CIL), cholesterol (chol), and mRNA. Yet the mechanism of pH-dependent response that is believed to cause endosomal release of LNPs is not well understood. Here, we show that eGFP (enhanced green fluorescent protein) protein expression in the mouse liver mediated by the ionizable lipids DLin-MC3-DMA (MC3), DLin-KC2-DMA (KC2), and DLinDMA (DD) ranks MC3 ≥ KC2 > DD despite similar delivery of mRNA per cell in all cell fractions isolated. We hypothesize that the three CIL-LNPs react differently to pH changes and hence study the structure of CIL/chol bulk phases in water. Using synchrotron X-ray scattering a sequence of ordered CIL/chol mesophases with lowering pH values are observed. These phases show isotropic inverse micellar, cubic Fd3m inverse micellar, inverse hexagonal and bicontinuous cubic Pn3m symmetry. If polyadenylic acid, as mRNA surrogate, is added to CIL/chol, excess lipid coexists with a condensed nucleic acid lipid phase. The next-neighbor distance in the excess phase shows a discontinuity at the Fd3m inverse micellar to inverse hexagonal transition occurring at pH 6 with distinctly larger spacing and hydration for DD vs. MC3 and KC2. In mRNA LNPs, DD showed larger internal spacing, as well as retarded onset and reduced level of DD-LNP-mediated eGFP expression in vitro compared to MC3 and KC2. Our data suggest that the pH-driven Fd3m- transition in bulk phases is a hallmark of CIL-specific differences in mRNA LNP efficacy.publishedVersio

Synthesis of photoactivable Pt(IV) prodrug loaded on NaYF4 based upconversion nanoparticles functionalized with 2-deoxy-D-glucose and its evaluation for targeted cancer therapy

Nano-formulation based on Tm, Yb doped NaYF4 upconversion nanoparticles (UCNPs) functionalized with 2-deoxy-D-glucose have been synthesized to load the photoactivable Pt(IV) prodrug, cis-[PtI2(NH3)2(OCOCH2CH2COOH)2]. The Pt(IV) prodrug has been synthesized by oxidation of cis-[PtI2(NH3)2] to [PtI2(OH)2(NH3)2] and its further treatment with succinic anhydride. It is loaded through ester bond formation between the carboxyl groups of Pt(IV) prodrug with hydroxyl groups of 2-deoxy-D-glucose (2-DG) coated on UCNPs. The cytotoxicity of formulation after exposing to 385 nm UV light and in absence of light is evaluated against MCF-7 cell lines by MTT assay. The results have revealed enhanced cytotoxicity of UV exposed nano-formulation. Additionally, the clonogenic assay has exhibited the decrease in plating efficiency as inferred from decreased surviving fraction around 20% only for UV activated formulation as compared to formulation in dark, as well as merely Pt(IV) prodrug. These results are indicative that more internalization of the formulation inside the cancer cells was achieved due to the presence of 2-DG rendering more efficiency to kill cancer cells

Estimation of tuberculosis incidence at subnational level using three methods to monitor progress towards ending TB in India, 2015–2020

Objectives We verified subnational (state/union territory (UT)/district) claims of achievements in reducing tuberculosis (TB) incidence in 2020 compared with 2015, in India.Design A community-based survey, analysis of programme data and anti-TB drug sales and utilisation data.Setting National TB Elimination Program and private TB treatment settings in 73 districts that had filed a claim to the Central TB Division of India for progress towards TB-free status.Participants Each district was divided into survey units (SU) and one village/ward was randomly selected from each SU. All household members in the selected village were interviewed. Sputum from participants with a history of anti-TB therapy (ATT), those currently experiencing chest symptoms or on ATT were tested using Xpert/Rif/TrueNat. The survey continued until 30 Mycobacterium tuberculosis cases were identified in a district.Outcome measures We calculated a direct estimate of TB incidence based on incident cases identified in the survey. We calculated an under-reporting factor by matching these cases within the TB notification system. The TB notification adjusted for this factor was the estimate by the indirect method. We also calculated TB incidence from drug sale data in the private sector and drug utilisation data in the public sector. We compared the three estimates of TB incidence in 2020 with TB incidence in 2015.Results The estimated direct incidence ranged from 19 (Purba Medinipur, West Bengal) to 1457 (Jaintia Hills, Meghalaya) per 100 000 population. Indirect estimates of incidence ranged between 19 (Diu, Dadra and Nagar Haveli) and 788 (Dumka, Jharkhand) per 100 000 population. The incidence using drug sale data ranged from 19 per 100 000 population in Diu, Dadra and Nagar Haveli to 651 per 100 000 population in Centenary, Maharashtra.Conclusion TB incidence in 1 state, 2 UTs and 35 districts had declined by at least 20% since 2015. Two districts in India were declared TB free in 2020