Efficient in vitro and in vivo transfection of self-amplifying mRNA with linear poly(propylenimine) and poly(ethylenimine-propylenimine) random copolymers as non-viral carriers.

Messenger RNA (mRNA) based vaccines have been introduced worldwide to combat the Covid-19 pandemic. These vaccines consist of non-amplifying mRNA formulated in lipid nanoparticles (LNPs). Consequently, LNPs are considered benchmark non-viral carriers for nucleic acid delivery. However, the formulation and manufacturing of these mRNA-LNP nanoparticles are expensive and time-consuming. Therefore, we used self-amplifying mRNA (saRNA) and synthesized novel polymers as alternative non-viral carrier platform to LNPs, which enable a simple, rapid, one-pot formulation of saRNA-polyplexes. Our novel polymer-based carrier platform consists of randomly concatenated ethylenimine and propylenimine comonomers, resulting in linear, poly(ethylenimine-ran-propylenimine) (L-PEIx-ran-PPIy) copolymers with controllable degrees of polymerization. Here we demonstrate in multiple cell lines, that our saRNA-polyplexes show comparable to higher in vitro saRNA transfection efficiencies and higher cell viabilities compared to formulations with Lipofectamine MessengerMAX™ (LFMM), a commercial, lipid-based carrier considered to be the in vitro gold standard carrier. This is especially true for our in vitro best performing saRNA-polyplexes with N/P 5, which are characterised with a size below 100 nm, a positive zeta potential, a near 100% encapsulation efficiency, a high retention capacity and the ability to protect the saRNA from degradation mediated by RNase A. Furthermore, an ex vivo hemolysis assay with pig red blood cells demonstrated that the saRNA-polyplexes exhibit negligible hemolytic activity. Finally, a bioluminescence-based in vivo study was performed over a 35-day period, and showed that the polymers result in a higher and prolonged bioluminescent signal compared to naked saRNA and L-PEI based polyplexes. Moreover, the polymers show different expression profiles compared to those of LNPs, with one of our new polymers (L-PPI250) demonstrating a higher sustained expression for at least 35 days after injection.

Linear Poly(ethylenimine-propylenimine) Random Copolymers for Gene Delivery: From Polymer Synthesis to Efficient Transfection with High Serum Tolerance.

Naturally occurring oligoamines, such as spermine, spermidine, and putrescine, are well-known regulators of gene expression. These oligoamines frequently have short alkyl spacers with varying lengths between the amines. Linear polyethylenimine (PEI) is a polyamine that has been widely applied as a gene vector, with various formulations currently in clinical trials. In order to emulate natural oligoamine gene regulators, linear random copolymers containing both PEI and polypropylenimine (PPI) repeat units were designed as novel gene delivery agents. In general, statistical copolymerization of 2-oxazolines and 2-oxazines leads to the formation of gradient copolymers. In this study, however, we describe for the first time the synthesis of near-ideal random 2-oxazoline/2-oxazine copolymers through careful tuning of the monomer structures and reactivity as well as polymerization conditions. These copolymers were then transformed into near-random PEI-PPI copolymers by controlled side-chain hydrolysis. The prepared PEI-PPI copolymers formed stable polyplexes with GFP-encoding plasmid DNA, as validated by dynamic light scattering. Furthermore, the cytotoxicity and transfection efficiency of polyplexes were evaluated in C2C12 mouse myoblasts. While the polymer chain length did not significantly increase the toxicity, a higher PPI content was associated with increased toxicity and also lowered the amount of polymers needed to achieve efficient transfection. The transfection efficiency was significantly influenced by the degree of polymerization of PEI-PPI, whereby longer polymers resulted in more transfected cells. Copolymers with 60% or lower PPI content exhibited a good balance between high plasmid-DNA transfection efficiency and low toxicity. Interestingly, these novel PEI-PPI copolymers revealed exceptional serum tolerance, whereby transfection efficiencies of up to 53% of transfected cells were achieved even under 50% serum conditions. These copolymers, especially PEI-PPI with DP500 and a 1:1 PEI/PPI ratio, were identified as promising transfection agents for plasmid DNA.

Analgesic effects of preinjection low-level laser/light therapy (LLLT) before third molar surgery: a double-blind randomized controlled trial.

OBJECTIVE: The aim of this study was to evaluate the analgesic effects of low-level laser therapy (LLLT) on preinjection sites in patients scheduled for third molar removal. STUDY DESIGN: This double-blind randomized controlled trial included 163 healthy patients undergoing third molar extractions. The study participants were randomly divided into an LLLT and a placebo group. Objective and subjective data sets were obtained from physiologic feedback (heart rate and sweat response) and a questionnaire, respectively. In the LLLT group, each targeted injection site was irradiated twice with 198 mW continuous wave for 30 seconds with a 0.088 cm2 focal spot at an applied energy of 5.94 J and fluence of 67.50 J/cm2. Measurements were recorded from 4 time-points during data acquisition. RESULTS: There was no significant difference between the LLLT and placebo groups in pain experience scores associated with the injected sites for maxillary or mandibular third molar extractions. Mean heart rates before and during injection were lower in the LLLT group than in the placebo group for both maxillary and mandibular regions. No statistically significant differences were observed for any remaining parameters. CONCLUSIONS: The present data indicated that preinjection LLLT did not effectively decrease the pain felt during local anesthetic injections before third molar surgery.

Self-elimination of risk factors for recurrent vaginal candidosis.

Women suffering from recurrent vulvo-vaginal candidosis (RVC) often follow medical and non-medical advices to diminish the severity and frequency of the recurrences, but the impact of such interventions is unclear. The aim of this study was to identify differences in life style habits of women with RVC compared with normal women and to define which changes have influenced the frequency of recurrences in these women. Fifty-one women with RVC and 51 age-matched control women without a history of RVC were sent a questionnaire. History of allergic disease (OR 2.8) and use of corticoids (OR 5) were more frequent in patients with RVC than controls. When interrogated about beneficial changes introduced in their life style habits, lowering the intake of sugars, preventing perineum humidity and stopping contraceptive pills were factors offering substantial improvement. Apart from an increased risk of having an allergic constitution, no differences in the medical history or life style habits were evident between women with RVC and healthy women. However, women with RVC have introduced several changes in life style habits that proved beneficial to them. Among these changes, lowering intake of sugars, preventing perineum humidity and stopping oral contraceptives were the most important.

IGRT in rectal cancer.

To date, no great interest has been shown in the clinical implementation of recent Image-guided radiation therapy (IGRT) modalities in rectal cancer since only a few studies have been published on this issue. This may be explained by the fact that with current treatment modalities locoregional recurrences are already very low (around 10%). However, there is still room for improvement in treatment of high risk patients (cT3 CRM+, cT4, N+). In these patients better results may be obtained improving radiation technique from 2D to 3D, which showed to be more reliable in terms of target coverage. Also, when higher doses are delivered, Intensity Modulated Radiation Therapy (IMRT) may be used to spare small bowel. But before employing 3D irradiation or IMRT, a proper definition of our clinical target volume (CTV) and planning target volume (PTV) is needed. The CTV should encompass the tumour site, the mesorectum and the lateral nodes, recognized as the most likely sites of local recurrence, with different incidence according to tumour stage. Recent studies discussed the correct delineation of these target volumes in respect of tumour site and stage. From the preliminary results of a study conducted in Rome University 2D planning seemed insufficient to cover the different target volumes especially in T4 patients compared to 3D planning. Also an appropriate PTV margin is necessary in order to manage set-up errors and organ motion. Particularly in these patients, the knowledge of mesorectal movement is required to avoid target missing. Large mesorectal displacements were observed in a study carried out in Leuven University in collaboration with Rome University. A systematic review of the literature together with the data from these first experiences led to the awareness that IGRT could help us to follow the target volume and organs at risk during the treatment, allowing adjustments to improve accuracy in dose delivery, especially when dose escalation studies are planned in the treatment of rectal cancer.

The use of magnetic sensors to monitor moderate deep inspiration breath hold during breast irradiation with dynamic MLC compensators.

BACKGROUND AND PURPOSE: To reduce the dose to the heart during left breast irradiation, a moderate deep breath hold technique (MDIBH) was introduced. Originally, verification of the MDIBH was performed with portal images acquired in movie loop during the treatment delivery. However, this verification method is not compatible with the use of dynamic MLC compensation, recently introduced because of its often superior dose distribution. Magnetic sensors were evaluated as an additional/alternative method to monitor the breath hold. MATERIAL AND METHODS: In a first phase, the reproducibility of MDIBH for breast patients was evaluated by investigating for 19 patients the set-up errors derived from portal images in cine loop acquisition during MDIBH. In a second phase, for 10 patients, the breathing curves recorded by magnetic sensors were used to monitor beam-on and beam-off while portal images were simultaneously recorded in movie loop. In a third phase, breast patients treated with dynamic MLC compensation were trained for MDIBH and monitored with magnetic sensors. RESULTS: The interfraction reproducibility of MDIBH for the initial 19 patients was recorded: the mean set-up error, the systematic and the random deviations are all smaller than 4mm in the anterior-posterior direction and in the cranio-caudal direction and smaller than 2 degrees along the rotation axis. Magnetic sensors provided a reproducible breathing curve: while the mean amplitude recorded for 10 patients varied substantially between patients, the individual standard deviation of the amplitude for each session was smaller than 3mm. For these 10 patients, the intrafraction set-up variation between the first portal image of two consecutive breath holds and the intra-breath hold set-up variation between the first and last portal image of each breath hold is smaller than 2mm in the anterior-posterior direction, smaller than 3mm in the cranio-caudal direction and smaller than 1.5 degrees along the rotation axis. CONCLUSION: Using magnetic sensors to record the breathing curve of left breast patients in MDIBH, a verification method was developed, suitable for combining MDIBH with dynamic MLC compensation.