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Control over the agglomeration state of manufactured particle systems for drug and oligonucleotide intracellular delivery is paramount to ensure reproducible and scalable therapeutic efficacy. Ultrasonication is a well-established mechanism for the deagglomeration of bulk powders in dispersion. Its use in manufacturing requires strict control of the uniformity and reproducibility of the cavitation field within the sample volume to minimise within-batch and batch-to-batch variability. In this work, we demonstrate the use of a reference cavitating vessel which provides stable and reproducible cavitation fields over litre-scale volumes to assist the controlled deagglomeration of a novel non-viral particle-based plasmid delivery system. The system is the Nuvec delivery platform, comprising polyethylenimine-coated spiky silica particles with diameters of ∼ 200 nm. We evaluated the use of controlled cavitation at different input powers and stages of preparation, for example before and after plasmid loading. Plasmid loading was confirmed by X-ray photoelectron spectroscopy and gel electrophoresis. The latter was also used to assess plasmid integrity and the ability of the particles to protect plasmid from potential degradation caused by the deagglomeration process. We show the utility of laser diffraction and differential centrifugal sedimentation in quantifying the efficacy of product de-agglomeration in the microscale and nanoscale size range respectively. Transmission electron microscopy was used to assess potential damages to the silica particle structure due to the sonication process., Competing Interests: Declaration of Competing Interest 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., (Copyright © 2022 The Author(s). Published by Elsevier B.V. All rights reserved.)

Patients with stable vitiligo can be helped surgically using transplantation of autologous cultured melanocytes, but there is a need for a culture methodology that is free from xenobiotic agents and for a simple way of delivering cultured melanocytes to the patient to achieve pigmentation with good wound healing. The aim of this study was to develop a chemically defined surface, suitable for the co-culture of melanocytes and keratinocytes which could be used in the future for the treatment vitiligo patients to achieve both restoration of pigmentation and good wound healing. Two keratinocyte growth media and two melanocyte growth media were compared; two of these were serum free. Cells were seeded on a range of chemically defined substrates (produced by plasma polymerisation of acrylic acid, allylamine or a mixture of these monomers) either as mono- or co-cultures. Melanocytes and keratinocytes attached and proliferated on both acid and amine substrates (without significant preferences), and co-cultures of cells proliferated more successfully than individual cultures. One media, M2, which is serum free, supported expansion of melanocytes and to a lesser extent keratinocytes on several plasma polymer substrates. In conclusion, these data indicate that a combination of a chemically defined substrate with M2 media allows serum-free co-culture of melanocytes and keratinocytes.

Ultra-violet Ozone (UVO) modified polystyrene (PS) surfaces were analyzed by X-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM), contact angle (CA), optical microscopy (OM) and cell culture experiments. UV/Ozone treatment up to 900 s was used to increase the surface oxygen concentration of PS surfaces from 0% to approximately 35% (unwashed) and 0% to approximately 27% (washed). The observed differences in oxygen concentration, between washed and unwashed surfaces, have been previously attributed to the removal of low molecular weight debris produced in this treatment process. Surface roughness (Rq) is known to affect cellular attachment and proliferation. AFM studies of the UV/Ozone treated PS surfaces show the surface roughness is an order of magnitude less than that expected to cause an effect. UV/Ozone treatment of PS showed a marked change in CA which decreased to approximately 60 degrees after 900 s treatment. The increased attachment and proliferation of Chinese hamster ovarian (CHO) and mouse embryo 3T3-L1 (3T3) cells on the treated surfaces compared to untreated PS were found to correlate strongly with the increase in surface oxygen concentration. Surface chemical oxidation patterns on the PS were produced using a simple masking technique and a short UV/Ozone treatment time, typically 20-45 s. The chemical patterns on PS were visualized by water condensation and the spatially selective attachment of CHO and 3T3-L1 cells cultured with 10% (v/v) serum. This paper describes an easily reproducible, one step technique to produce a well-defined, chemically heterogeneous surface with a cellular resolution using UV/Ozone modification. By using a variety of cell types, that require different media conditions, we have been able to expand the potential applications of this procedure.