Natural photosensitizers, such as curcumin or parietin, play a vital role in photodynamic therapy (PDT), causing a light-mediated reaction that kills cancer cells. PDT is a promising treatment option for glioblastoma, especially when combined with nanoscale drug delivery systems. The curcumin- or parietin-loaded lipid nanoparticles were prepared via dual asymmetric centrifugation and subsequently characterized through physicochemical analyses including dynamic light scattering, laser Doppler velocimetry, and atomic force microscopy. The combination of PDT and lipid nanoparticles has been evaluated in vitro regarding uptake, safety, and efficacy. The extensive and well-vascularized chorioallantois membrane (CAM) of fertilized hen\'s eggs offers an optimal platform for three-dimensional cell culture, which has been used in this study to evaluate the photodynamic efficacy of lipid nanoparticles against glioblastoma cells. In contrast to other animal models, the CAM model lacks a mature immune system in an early stage, facilitating the growth of xenografts without rejection. Treatment of xenografted U87 glioblastoma cells on CAM was performed to assess the effects on tumor viability, growth, and angiogenesis. The xenografts and the surrounding blood vessels were targeted through topical application, and the effects of photodynamic therapy have been confirmed microscopically and via positron emission tomography and X-ray computed tomography. Finally, the excised xenografts embedded in the CAM were analyzed histologically by hematoxylin and eosin and KI67 staining.

Thermoresponsive drug delivery systems have been used to treat diseases that cause hyperthermia or elevated body tissue temperatures, viz., rheumatoid arthritis and different cancers. The aim of the study was to enhance berberine (BER) release using thermosensitive nanostructured lipid carriers (TNLCs) through intra-articular administration for the management of arthritis. TNLCs were prepared using binary mixtures of stearic acid and decanoic acid as solid and liquid lipids, respectively. Lipid mixtures with an optimum melting point were assessed using differential scanning calorimetry studies. In vitro characterization of the BER TNLCs included particle size, zeta potential, entrapment efficiency, and drug release at 37 °C and 41 °C. Joint diameter measurement, real-time polymerase chain reaction (RT-PC) analysis, enzyme-linked immunosorbent assay (ELISA) for inflammatory markers, and histological evaluation of the dissected joints were all performed in vivo on rats with adjuvant-induced arthritis. In vitro characterization revealed negatively charged BER-loaded TNLCs with a spherical shape, particle size less than 500 nm, BER entrapment efficiency up to 79%, and a high drug release rate at an elevated temperature of 41 °C. In silico studies revealed the affinity of BER to different formula components and to the measured biomarkers. In vivo assessment of the optimum TNLCs showed that BER TNLCs were superior to the BER solution suspension regarding their effect on inflammatory biomarkers, joint diameter, and histological studies.

The synthesis of nanoparticles in microchannels promises the advantages of small size, uniform shape and narrow size distribution. However, only with insights into the mixing processes can the most suitable designs and operating conditions be systematically determined. Coaxial lamination mixers (CLM) built by 2-photon polymerization can operate long-term stable nanoparticle precipitation without fouling issues. Contact of the organic phase with the microchannel walls is prevented while mixing with the aqueous phase is intensified. A coaxial nozzle allows 3D hydrodynamic focusing followed by a sequence of stretch-and-fold units. By means of a digital twin based on computational fluid dynamics (CFD) and numerical evaluation of mixing progression, the influences of operation conditions are now studied in detail. As a measure for homogenization, the mixing index (MI) was extracted as a function of microchannel position for different operating parameters such as the total flow rate and the share of solvent flow. As an exemplary result, behind a third stretch-and-fold unit, practically perfect mixing (MI>0.9) is predicted at total flow rates between 50 µL/min and 400 µL/min and up to 20% solvent flow share. Based on MI values, the mixing time, which is decisive for the size and dispersity of the nanoparticles, can be determined. Under the conditions considered, it ranges from 5 ms to 54 ms. A good correlation between the predicted mixing time and nanoparticle properties, as experimentally observed in earlier work, could be confirmed. The digital twin combining CFD with the MI methodology can in the future be used to adjust the design of a CLM or other micromixers to the desired total flow rates and flow rate ratios and to provide valuable predictions for the mixing time and even the properties of nanoparticles produced by microfluidic antisolvent precipitation.

Lipid nanoparticles (LNPs) are currently having an increasing impact on nanomedicines as delivery agents, among others, of RNA molecules (e.g., short interfering RNA for the treatment of hereditary diseases or messenger RNA for the development of COVID-19 vaccines). Despite this, the delivery of plasmid DNA (pDNA) by LNPs in preclinical studies is still unsatisfactory, mainly due to the lack of systematic structural and functional studies on DNA-loaded LNPs. To tackle this issue, we developed, characterized, and tested a library of 16 multicomponent DNA-loaded LNPs which were prepared by microfluidics and differed in lipid composition, surface functionalization, and manufacturing factors. 8 out of 16 formulations exhibited proper size and zeta potential and passed to the validation step, that is, the simultaneous quantification of transfection efficiency and cell viability in human embryonic kidney cells (HEK-293). The most efficient formulation (LNP15) was then successfully validated both in vitro, in an immortalized adult keratinocyte cell line (HaCaT) and in an epidermoid cervical cancer cell line (CaSki), and in vivo as a nanocarrier to deliver a cancer vaccine against the benchmark target tyrosine-kinase receptor HER2 in C57BL/6 mice. Finally, by a combination of confocal microscopy, transmission electron microscopy and synchrotron small-angle X-ray scattering, we were able to show that the superior efficiency of LNP15 can be linked to its disordered nanostructure consisting of small-size unoriented layers of pDNA sandwiched between closely apposed lipid membranes that undergo massive destabilization upon interaction with cellular lipids. Our results provide new insights into the structure-activity relationship of pDNA-loaded LNPs and pave the way to the clinical translation of this gene delivery technology.

Breast cancer is a complex heterogeneous disease with unique molecular subtypes, which limits the development of optimized treatment strategies for each subtype. Cancer gene therapy and potential therapeutics for advanced/refractory cancers can be promising for breast cancer. Combining tumor-tropic lipid nanoparticles (LNPs) and inducible caspase-9 (iC9) mRNA, we aimed to develop a novel treatment strategy for refractory breast cancer. LNP\'s anti-tumor effects were tested in vitro in three breast cancer cell lines: MDA-MB231, SKBR3, and MCF-7. Tumor cells were treated with LNPs encapsulated with eGFP or iC9 mRNA and chemical inducers of dimerization (CID). Apoptosis-related genes were evaluated by reverse transcriptase quantitative PCR. LNPs could efficiently deliver encapsulated GFP mRNA to all three cancer cell lines (>80% GFP expression. in target cells). Furthermore, LNPs encapsulated with iC9 mRNA (iC9-LNPs) and CID showed cytotoxic activity against all cancer cell lines in vitro. Interestingly, susceptibility to iC9 gene therapy was heterogeneous among cancer cell lines. iC9-LNPs with CID-induced potent cytotoxic effects against SKBR3 and MDA-MB231 cells, but only a mild cytotoxic effect on MCF7 cells. Quantification of apoptosis-related genes suggested that a high BAX/Bcl-2 ratio might be associated with iC9-LNP + CID susceptibility. Thus, cancer gene therapy using iC9-LNPs and CID could be a promising alternative for the treatment of breast cancers, especially for aggressive breast cancers.

Surface charge polarity and density influence the immune clearance and cellular uptake of intravenously administered lipid nanoparticles (LNPs), thus determining the efficiency of their delivery to the target. Here, we modified the surface charge with ascorbyl palmitate (AsP) used as a negatively charged lipid. AsP-PC-LNPs were prepared by dispersion and ultrasonication of AsP and phosphatidylcholine (PC) composite films at various ratios. AsP inserted into the PC film with its polar head outward. The pKa for AsP was 4.34, and its ion form conferred the LNPs with negative surface charge. Zeta potentials were correlated with the amount and distribution of AsP on the LNPs surface. DSC, Raman and FTIR spectra, and molecular dynamics simulations disclosed that AsP distributed homogeneously in PC at 1−8% (w/w), and there were strong hydrogen bonds between the polar heads of AsP and PC (PO2−), which favored LNPs’ stability. But at AsP:PC > 8% (w/w), the excessive AsP changed the interaction modes between AsP and PC. The AsP−PC composite films became inhomogeneous, and their phase transition behaviors and Raman and FTIR spectra were altered. Our results clarified the mechanism of surface charge modification by AsP and provided a rational use of AsP as a charged lipid to modify LNP surface properties in targeted drug delivery systems. Furthermore, AsP−PC composites were used as phospholipid-based biological membranes to prepare paclitaxel-loaded LNPs, which had stable surface negative charge, better tumor targeting and tumor inhibitory effects.

OBJECTIVE: The roll-out of the Pfizer-BioNTech BNT162b2 COVID-19 vaccine has brought many logistical challenges, such as the absence of comprehensive stability data leading to strict handling instructions during dilution and administration. Accidental mishandling therefore presents challenging clinical dilemmas, which often led vaccine providers to err on the side of caution and discard mishandled vials rather than risk administering ineffective vaccine. This study aims to answer key questions about the vaccine\'s stability to allow for a more informed decision-making process should a non-conformity occur.
METHODS: Residual vaccine in freshly used, but appropriately stored vials collected from vaccination centres in Brighton, UK, were tested after exposure to various handling conditions and analysed by dynamic light scattering to determine the size of the lipid-mRNA nanoparticles, and gel electrophoresis to visualise the mRNA integrity and separation from the lipid formulation.
RESULTS: Knocking or dropping vaccine samples from small heights resulted in lowest levels of instability, indicating low risk of compromising clinical efficacy. However, repeated drawing and injecting through 23 G needles at high speed and, more significantly, shaking and vortexing led to progressive increase in the size and polydispersity index of the lipid-mRNA nanoparticles, coupled with or caused by up to ~50% release of mRNA from the lipid formulation. This is thought to impact the vaccine\'s efficacy due to lack of free mRNA protection and cellular internalisation.
CONCLUSIONS: These results reiterate the importance of adhering to the manufacturer\'s instructions on handling, especially with regard to shaking and exposing the vaccine to excessive vibration.

Coronavirus (SARS-CoV-2) as a global pandemic has attracted the attention of many scientific centers to find the right treatment. We expressed and purified the recombinant receptor-binding domain (RBD) of the SARS-CoV-2 spike (S) protein, and specific RBD aptamers were designed using SELEX method. RNAi targeting nucleocapsid phosphoprotein was synthesized and human lung cells were inoculated with aptamer-functionalized lipid nanoparticles (LNPs) containing RNAi. The results demonstrated that RBD aptamer having KD values of 0.290 nm possessed good affinity. Based on molecular docking and efficacy prediction analysis, siRNA molecule was showed the best action. LNPs were appropriately functionalized by aptamer and contained RNAi molecules. Antiviral assay using q-PCR and ELISA demonstrated that LNP functionalized with 35 µm Apt and containing 30 nm RNAi/ml of cell culture had the best antiviral activity compared to other concentrations. Applied aptamer in the nanocarrier has two important functions. First, it can deliver the drug (RNAi) to the surface of epithelial cells. Second, by binding to the SARS-CoV-2 spike protein, it inhibits the virus entrance into cells. Our data reveal an interaction between the aptamer and the virus, and RNAi targeted the virus RNA. CT scan and the clinical laboratory tests in a clinical case study, a 36-year old man who presented with severe SARS-CoV-2, demonstrated that inhalation of 10 mg Apt-LNPs-RNAi nebulized/day for six days resulted in an improvement in consolidation and ground-glass opacity in lungs on the sixth day of treatment. Our findings suggest the treatment of SARS-CoV-2 infection through inhalation of Aptamer-LNPs-RNAi.

Lipid nanoparticles are a successful carrier system for dermal drug delivery. They possess various beneficial properties, i.e., increased chemical stability for chemically labile compounds, increased dermal penetration of active compounds, or skin carrying properties after dermal application due to the formation of a so-called \"invisible patch.\" Despite manifold studies showing these properties individually, a study that investigates if one lipid nanoparticle formulation can really combine all the above-mentioned benefits at once is not yet available. In the present study, lipid nanoparticles (NLC) were produced and characterized regarding their physico-chemical properties. The chemical stability of the incorporated active ingredient (AI) was determined, as well as the dermal penetration efficacy of the AI, and the skin carrying properties of the NLC after dermal penetration. The properties of the NLC were compared to classical formulations, i.e., AI dissolved in pure oil, an o/w cream base and a nanoemulsion. All formulations contained similar lipids and emulsifiers, which allowed for a direct comparison of the different properties. NLC were shown to provide most efficient chemical stabilization and most efficient dermal penetration for the AI. The formation of the invisible patch was shown for the NLC but not for the other formulations. Skin hydration and skin carrying properties were also most pronounced for the NLC. Results provide evidence that NLC can combine all beneficial effects that were previously described in one formulation. Thus, providing evidence that NLC are a holistically superior formulation principle when compared to other formulation principles.

In a first-in-human study immune responses to rabies virus glycoprotein (RABV-G)-mRNA vaccine were dependent on the route of administration, necessitating specialized devices. Following successful preclinical studies with mRNA encapsulated in lipid nanoparticles (LNP), we tested an mRNA-LNP formulation (CV7202).
In this phase 1, multi-center, controlled study in Belgium and Germany we enrolled 55 healthy 18-40-year-olds to receive intramuscular injections of 5 μg (n = 10), 1 μg (n = 16), or 2 μg (n = 16) CV7202 on Day 1; subsets (n = 8) of 1 μg and 2 μg groups received second doses on Day 29. Controls (n = 10) received rabies vaccine, Rabipur, on Days 1, 8 and 29. Safety and reactogenicity were assessed up to 28 days post-vaccination using diary cards; immunogenicity was measured as RABV-G-specific neutralizing titers (VNT) by RFFIT and IgG by ELISA.
As initially tested doses of 5 μg CV7202 elicited unacceptably high reactogenicity we subsequently tested 1 and 2 μg doses which were better tolerated. No vaccine-related serious adverse events or withdrawals occurred. Low, dose-dependent VNT responses were detectable from Day 15 and by Day 29%, 31% and 22% of 1, 2 and 5 μg groups, respectively, had VNTs ≥ 0·5 IU/mL, considered an adequate response by the WHO. After two 1 or 2 μg doses all recipients had titers ≥ 0.5 IU/mL by Day 43. Day 57 GMTs were not significantly lower than those with Rabipur, which elicited adequate responses in all vaccinees after two doses. CV7202-elicited VNT were significantly correlated with RABV-G-specific IgG antibodies (r2 = 0.8319, p < 0.0001).
Two 1 μg or 2 μg doses of CV7202 were well tolerated and elicited rabies neutralizing antibody responses that met WHO criteria in all recipients, but 5 μg had unacceptable reactogenicity for a prophylactic vaccine. ClinicalTrials.gov Identifier: NCT03713086.