Células Natural Killer y cáncer

El cáncer es uno de los mayores problemas de salud pública global a día de hoy siendo, según datos del INE(Instituto Nacional de Estadística), la primera causa de muerte en los hospitales españoles. Múltiples frentes en investigación y tratamiento se encuentran abiertos actualmente y uno de ellos es la inmunoterapia con células natural killer (NK). En ella, mediante diferentes técnicas, se usan a las células NK para detectar y erradicar células tumorales. Pueden usarse bien como monoterapia o bien en combinación con otras células u otros fármacos, como los anticuerpos monoclonales

New multi-material formulations for vat-photopolymerisation and their application in manufacturing ocular implants

Additive Manufacturing (AM), often referred to as 3D printing, encompasses a series of processes that build structures in an additive fashion, layer by layer. This is usually possible by translating a 3D computer-aided design (CAD) into a standard tessellation language (STL) file that is then directly manufactured into a physical structure. The process that manufactures structures illuminating liquid photosensitive resins with a light source is called vat-photopolymerisation. From the different vat-photopolymerisation techniques, two-photon polymerisation is the technique that allows micro- and nano-printing at the highest resolution. Besides high resolution, it also offers the possibility of manufacturing complex structures without need for support. And it also allows to select degrees of porosity, defined shapes, or intricate topologies. These features had made the technique attractive for several applications like microfluidics, metamaterials, cell culture, or drug delivery. However, there are several challenges that need to be resolved for the technique to reach its full potential and be embraced on a commercial basis outside the research community. This thesis aims to tackle two of the disadvantages of this technique. First, the lack of suitable materials available for it. Currently, there are few materials that are 2PP processable that are also of interest for biological applications. Second, the research developed on the technique has mainly focused on mono-material formulations. Therefore, the work presented in this thesis addresses both drawbacks by developing new materials that could be potentially used for biological applications and optimises them for 2PP processing in multi-material formulations. This was achieved by initially synthesizing a new mono–functional poly(trimethylene carbonate) acrylate (PTMCA) polymer and optimizing it for 2PP printing in several multi-material formulations. The optimisation was done by mixing the biodegradable PTMCA with a hydrophilic (poly(ethylene glycol) diacrylate (PEGDA)) and hydrophobic (tricyclo [5.2.1.02,6] decanedimethanol diacrylate (TCDMDA)) cross-linker monomer at different concentrations. The multi-material formulations containing the mono–functional PTMCA were successfully processed in the 2PP system, and the structures manufactured with them showed a sharp increase in reacted vinyl groups (RVG) compared to structures manufactured with only commercial monomer (between 20% and 50% increase depending on the formulation used). The addition of PTMCA to the monomers also significantly expanded the polymerisation threshold of the formulations when compared to thresholds of only cross-linkers. The presence and disposition of both materials in structures manufactured with multi-material formulations were confirmed via Time-of-Flight Secondary Ion Mass Spectrometry (TOF – SIMS). PTMCA + PEGDA samples showed a continuous single phase. PTMCA + TCDMDA samples showed phase separation at certain concentrations. This phase separation could be controlled by the formulation concentration or the processing parameters of the 2PP system. The library of 2PP materials was also expanded by synthesizing two new hyperbranched polymers: Hyperbranched poly(PEGDA) (HBpPEGDA) and Hyperbranched poly(TCDMDA) (HBpTCDMDA). This would allow to manufacture with highly functionalised materials with a high molecular weight, compared to the mono-functional low molecular weight PTMCA. The hyperbranched materials were optimized for 2PP manufacturing in mono-material (HBpTCDMDA based) or multi-material (HBpPEGDA based) formulations. Structures manufactured with HBpTCDMDA also showed a sharp increase in RVG (>50 %) when compared to structures manufactured with TCDMDA monomer. The hyperbranched formulations also significantly broadened the polymerization threshold when compared to thresholds of formulations prepared with commercial PEGDA and TCDMDA monomers. The presence of both materials in multi-material formulations was confirmed via ToF-SIMS, with a single continuous phase. Scaffolds printed with HBpTCDMDA also showed high viability on LIVE/DEAD assay with mouse fibroblasts, indicating the potential biocompatibility of this material. Last, to show the potential of one of the formulations developed in this thesis for a biological application, one of the PTMCA + PEGDA mixtures was selected and further optimised to fabricate a proof-of-concept intravitreal implant for sustained release of fluocinolone acetonide (FA). Different implant designs based on a diamond lattice were created to study the influence of implant geometry on drug release. Printing optimisation was initiated in the 2PP system to determine the initial feasibility of lattice design printing at small scale while using small amounts of formulation, but micro projection stereolithography (PμSLA) was chosen as the technique for scale-up and final implant manufacturing due to faster printing times compared to 2PP while still maintaining adequate resolution. N – vinyl pyrrolidone (NVP) was added to the formulation to facilitate drug release from the implant matrix. The percent of NVP needed was optimised by studying the release of model drug Rhodamine B from Ultraviolet (UV) cast samples. The concentration that allowed for a sustained release of ~80 % of model drug over 39 days was 25 wt% NVP, which was the formulation selected to manufacture the FA intravitreal implants. Cylindrical lattice implants (1.85 ± 0.05 mm in length, 0.5 mm in diameter) loaded with 6.25 wt% of FA were manufactured via PμSLA using the optimised formulation containing 25 wt% NVP. They showed high cell viability and no cytotoxicity in in vitro assays with human corneal epithelial cells. All lattice implants showed ~60 % of cumulative drug release over 35 days. The profile of release varied depending on the geometry of the implant, indicating an influence of the pore size and surface area over release. The microstructure analysis of samples indicated some phase separation and a possible preference of the drug for Poly - vinyl pyrrolidone (PVP), which further facilitated the drug release from the polymer matrix when it dissolved into aqueous media

New multi-material formulations for vat-photopolymerisation and their application in manufacturing ocular implants

Additive Manufacturing (AM), often referred to as 3D printing, encompasses a series of processes that build structures in an additive fashion, layer by layer. This is usually possible by translating a 3D computer-aided design (CAD) into a standard tessellation language (STL) file that is then directly manufactured into a physical structure. The process that manufactures structures illuminating liquid photosensitive resins with a light source is called vat-photopolymerisation. From the different vat-photopolymerisation techniques, two-photon polymerisation is the technique that allows micro- and nano-printing at the highest resolution. Besides high resolution, it also offers the possibility of manufacturing complex structures without need for support. And it also allows to select degrees of porosity, defined shapes, or intricate topologies. These features had made the technique attractive for several applications like microfluidics, metamaterials, cell culture, or drug delivery. However, there are several challenges that need to be resolved for the technique to reach its full potential and be embraced on a commercial basis outside the research community. This thesis aims to tackle two of the disadvantages of this technique. First, the lack of suitable materials available for it. Currently, there are few materials that are 2PP processable that are also of interest for biological applications. Second, the research developed on the technique has mainly focused on mono-material formulations. Therefore, the work presented in this thesis addresses both drawbacks by developing new materials that could be potentially used for biological applications and optimises them for 2PP processing in multi-material formulations. This was achieved by initially synthesizing a new mono–functional poly(trimethylene carbonate) acrylate (PTMCA) polymer and optimizing it for 2PP printing in several multi-material formulations. The optimisation was done by mixing the biodegradable PTMCA with a hydrophilic (poly(ethylene glycol) diacrylate (PEGDA)) and hydrophobic (tricyclo [5.2.1.02,6] decanedimethanol diacrylate (TCDMDA)) cross-linker monomer at different concentrations. The multi-material formulations containing the mono–functional PTMCA were successfully processed in the 2PP system, and the structures manufactured with them showed a sharp increase in reacted vinyl groups (RVG) compared to structures manufactured with only commercial monomer (between 20% and 50% increase depending on the formulation used). The addition of PTMCA to the monomers also significantly expanded the polymerisation threshold of the formulations when compared to thresholds of only cross-linkers. The presence and disposition of both materials in structures manufactured with multi-material formulations were confirmed via Time-of-Flight Secondary Ion Mass Spectrometry (TOF – SIMS). PTMCA + PEGDA samples showed a continuous single phase. PTMCA + TCDMDA samples showed phase separation at certain concentrations. This phase separation could be controlled by the formulation concentration or the processing parameters of the 2PP system. The library of 2PP materials was also expanded by synthesizing two new hyperbranched polymers: Hyperbranched poly(PEGDA) (HBpPEGDA) and Hyperbranched poly(TCDMDA) (HBpTCDMDA). This would allow to manufacture with highly functionalised materials with a high molecular weight, compared to the mono-functional low molecular weight PTMCA. The hyperbranched materials were optimized for 2PP manufacturing in mono-material (HBpTCDMDA based) or multi-material (HBpPEGDA based) formulations. Structures manufactured with HBpTCDMDA also showed a sharp increase in RVG (>50 %) when compared to structures manufactured with TCDMDA monomer. The hyperbranched formulations also significantly broadened the polymerization threshold when compared to thresholds of formulations prepared with commercial PEGDA and TCDMDA monomers. The presence of both materials in multi-material formulations was confirmed via ToF-SIMS, with a single continuous phase. Scaffolds printed with HBpTCDMDA also showed high viability on LIVE/DEAD assay with mouse fibroblasts, indicating the potential biocompatibility of this material. Last, to show the potential of one of the formulations developed in this thesis for a biological application, one of the PTMCA + PEGDA mixtures was selected and further optimised to fabricate a proof-of-concept intravitreal implant for sustained release of fluocinolone acetonide (FA). Different implant designs based on a diamond lattice were created to study the influence of implant geometry on drug release. Printing optimisation was initiated in the 2PP system to determine the initial feasibility of lattice design printing at small scale while using small amounts of formulation, but micro projection stereolithography (PμSLA) was chosen as the technique for scale-up and final implant manufacturing due to faster printing times compared to 2PP while still maintaining adequate resolution. N – vinyl pyrrolidone (NVP) was added to the formulation to facilitate drug release from the implant matrix. The percent of NVP needed was optimised by studying the release of model drug Rhodamine B from Ultraviolet (UV) cast samples. The concentration that allowed for a sustained release of ~80 % of model drug over 39 days was 25 wt% NVP, which was the formulation selected to manufacture the FA intravitreal implants. Cylindrical lattice implants (1.85 ± 0.05 mm in length, 0.5 mm in diameter) loaded with 6.25 wt% of FA were manufactured via PμSLA using the optimised formulation containing 25 wt% NVP. They showed high cell viability and no cytotoxicity in in vitro assays with human corneal epithelial cells. All lattice implants showed ~60 % of cumulative drug release over 35 days. The profile of release varied depending on the geometry of the implant, indicating an influence of the pore size and surface area over release. The microstructure analysis of samples indicated some phase separation and a possible preference of the drug for Poly - vinyl pyrrolidone (PVP), which further facilitated the drug release from the polymer matrix when it dissolved into aqueous media

Personalised 3D Printed Medicines: Which Techniques and Polymers Are More Successful?

The interindividual variability is an increasingly global problem when treating patients from different backgrounds with diverse customs, metabolism, and necessities. Dose adjustment is frequently based on empirical methods, and therefore, the chance of undesirable side effects to occur is high. Three-dimensional (3D) Printed medicines are revolutionsing the pharmaceutical market as potential tools to achieve personalised treatments adapted to the specific requirements of each patient, taking into account their age, weight, comorbidities, pharmacogenetic, and pharmacokinetic characteristics. Additive manufacturing or 3D printing consists of a wide range of techniques classified in many categories but only three of them are mostly used in the 3D printing of medicines: printing-based inkjet systems, nozzle-based deposition systems, and laser-based writing systems. There are several drawbacks when using each technique and also the type of polymers readily available do not always possess the optimal properties for every drug. The aim of this review is to give an overview about the current techniques employed in 3D printing medicines, highlighting their advantages, disadvantages, along with the polymer and drug requirements for a successful printing. The major application of these techniques will be also discussed

A 12-gene pharmacogenetic panel to prevent adverse drug reactions: an open-label, multicentre, controlled, cluster-randomised crossover implementation study

© 2023Background: The benefit of pharmacogenetic testing before starting drug therapy has been well documented for several single gene–drug combinations. However, the clinical utility of a pre-emptive genotyping strategy using a pharmacogenetic panel has not been rigorously assessed. Methods: We conducted an open-label, multicentre, controlled, cluster-randomised, crossover implementation study of a 12-gene pharmacogenetic panel in 18 hospitals, nine community health centres, and 28 community pharmacies in seven European countries (Austria, Greece, Italy, the Netherlands, Slovenia, Spain, and the UK). Patients aged 18 years or older receiving a first prescription for a drug clinically recommended in the guidelines of the Dutch Pharmacogenetics Working Group (ie, the index drug) as part of routine care were eligible for inclusion. Exclusion criteria included previous genetic testing for a gene relevant to the index drug, a planned duration of treatment of less than 7 consecutive days, and severe renal or liver insufficiency. All patients gave written informed consent before taking part in the study. Participants were genotyped for 50 germline variants in 12 genes, and those with an actionable variant (ie, a drug–gene interaction test result for which the Dutch Pharmacogenetics Working Group [DPWG] recommended a change to standard-of-care drug treatment) were treated according to DPWG recommendations. Patients in the control group received standard treatment. To prepare clinicians for pre-emptive pharmacogenetic testing, local teams were educated during a site-initiation visit and online educational material was made available. The primary outcome was the occurrence of clinically relevant adverse drug reactions within the 12-week follow-up period. Analyses were irrespective of patient adherence to the DPWG guidelines. The primary analysis was done using a gatekeeping analysis, in which outcomes in people with an actionable drug–gene interaction in the study group versus the control group were compared, and only if the difference was statistically significant was an analysis done that included all of the patients in the study. Outcomes were compared between the study and control groups, both for patients with an actionable drug–gene interaction test result (ie, a result for which the DPWG recommended a change to standard-of-care drug treatment) and for all patients who received at least one dose of index drug. The safety analysis included all participants who received at least one dose of a study drug. This study is registered with ClinicalTrials.gov, NCT03093818 and is closed to new participants. Findings: Between March 7, 2017, and June 30, 2020, 41 696 patients were assessed for eligibility and 6944 (51·4 % female, 48·6% male; 97·7% self-reported European, Mediterranean, or Middle Eastern ethnicity) were enrolled and assigned to receive genotype-guided drug treatment (n=3342) or standard care (n=3602). 99 patients (52 [1·6%] of the study group and 47 [1·3%] of the control group) withdrew consent after group assignment. 652 participants (367 [11·0%] in the study group and 285 [7·9%] in the control group) were lost to follow-up. In patients with an actionable test result for the index drug (n=1558), a clinically relevant adverse drug reaction occurred in 152 (21·0%) of 725 patients in the study group and 231 (27·7%) of 833 patients in the control group (odds ratio [OR] 0·70 [95% CI 0·54–0·91]; p=0·0075), whereas for all patients, the incidence was 628 (21·5%) of 2923 patients in the study group and 934 (28·6%) of 3270 patients in the control group (OR 0·70 [95% CI 0·61–0·79]; p <0·0001). Interpretation: Genotype-guided treatment using a 12-gene pharmacogenetic panel significantly reduced the incidence of clinically relevant adverse drug reactions and was feasible across diverse European health-care system organisations and settings. Large-scale implementation could help to make drug therapy increasingly safe. Funding: European Union Horizon 2020