Poly(3-hydroxybutyrate): A potential biodegradable excipient for direct 3D printing of pharmaceuticals

During the past decades, 3D printing has revolutionised different areas of research. Despite the considerable progress achieved in 3D printing of pharmaceuticals, the limited choice of suitable materials remains a challenge to overcome. The growing search for sustainable excipients has led to an increasing interest in biopolymers. Poly(3-hydroxybutyrate) (PHB) is a biocompatible and biodegradable biopolymer obtained from bacteria that could be efficiently employed in the pharmaceutical field. Here we aimed to demonstrate its potential application as a thermoplastic material for personalised medicine through 3D printing. More specifically, we processed PHB by using direct powder extrusion, a one-step additive manufacturing technique. To assess and denote the feasibility and versatility of the process, a 3D square model was manufactured in different dimensions (sidexheight: 12x2 mm; 18x2 mm; 24x2 mm) and loaded with increasing percentages of a model drug (up to 30% w/w). The manufacturing process was influenced by the drug content, and indeed, an increase in the amount of the drug determined a reduction in the printing temperature, without affecting the other parameters (such as the layer height). The composition of the model squares was investigated using Fourier-transform infrared spectroscopy, the resulting spectra confirmed that the starting materials were successfully incorporated into the final formulations. The thermal behaviour of the printed systems was characterized by differential scanning calorimetry, and thermal gravimetric analysis. Moreover, the sustained drug release profile of the formulations was performed over 21 days and showed to be dependent on the dimensions of the printed object and on the amount of loaded drug. Indeed, the formulation with 30% w/w in the dimension 24x2 mm released the highest amount of drug. Hence, the results suggested that PHB and direct powder extrusion technique could be promising tools for the manufacturing of prolonged release and personalised drug delivery forms

Chaperone-Assisted Soluble Expression of a Humanized Anti-EGFR ScFv Antibody in E. Coli

Purpose: Formation of inclusion bodies is a considerable obstacle threatening the advantages of E. coli expression system to serve as the most common and easiest system in recombinant protein production. To solve this problem, several strategies have been proposed among which application of molecular chaperones is of remarkable consideration. The aim of this study was to evaluate the effects of molecular chaperones on soluble expression of aggregation-prone humanized single chain antibody. Methods: To increase the solubility of a humanized single chain antibody (hscFv), different chaperone plasmids including PG-tf2 (GroES- GroEL- tig), ptf16 (tig) and pGro7 (GroES- GroEL) were co-expressed in BL21 cells containing pET-22b- hscFv construct. The solubility of recombinant hscFv was analyzed by SDS-PAGE. After purification of soluble hscFv by Ni-NTA column, the biological activity and cytotoxicity of the recombinant protein were tested by ELISA and MTT assay, respectively. Results: SDS-PAGE analysis of the hscFv revealed that chaperone utility remarkably increased (up to 50%) the solubility of the protein. ELISA test and MTT assay analyses also confirmed the biological activity of the gained hscFv in reaction with A431 cells (OD value: 2.6) and inhibition of their proliferation, respectively. Conclusion: The results of this study revealed that co-expression of chaperones with hscFv leads to remarkable increase in the solubility of the recombinant hscFv, which could be of great consideration for large scale production of recombinant single chain antibodies

An experimental framework for manufacturing nanomedicine via custom-made 3D-printed microfluidics

Nanoparticles offer a paradigm shift in therapeutic strategies. These structures, derived from diverse materials, exhibit unique properties such as enhanced drug delivery, controlled release, and targeted therapies. Microfluidic methods, a cutting-edge technology, revolutionize the fabrication of nanoparticles by enabling precise manipulation of fluids in microchannels. This approach provides advantages like scalable production, rapid mixing, and controlled physicochemical properties. Together, nanoparticles and microfluidics redefine therapeutic approaches. For instance, the collaboration has been exemplified in the production of lipid nanoparticles encapsulating mRNA vaccines for cancer and COVID-19, showcasing a transformative leap in the field of medicine. This convergence of nanotechnology and microfluidics holds immense promise for tailored and efficient drug delivery systems, maximizing treatment efficacy while minimizing side effects. In this thesis, the result of the conducted research on nanoparticle preparation utilizing the microfluidic method and Design of Experiments (DoE) during my PhD program has been presented. Initial projects delve into the investigation of novel applications in anticancer drug delivery. Leveraging carbohydrate targeting and the unique properties of berberine, innovative liposomal formulations demonstrate enhanced anti-proliferative effects. In addressing cutaneous mycoses, the thesis introduces keratin nanoparticles as a promising antifungal drug carrier. Tannic acid (TA) crosslinking proves superior to conventional crosslinkers, ensuring stability and effectiveness in tioconazole-loaded keratin nanoparticles (TCZ-KNP). The study exemplifies a tailored approach to combat topical fungal infections. Our third study aims to assess the impact of various nanomedicines on hematological malignancies (HM), offering new possibilities for enhanced patient outcomes, where curative treatments are scarce. Conventional top-down nanomedicine development methods in preclinical research are time-consuming, motivating a bottom-up microfluidic and DoE approach to expedite production, evaluation, and prediction of a nanoparticle (NP) library composed of liposomes, lipid nanoparticles (LNP), and nanoemulsions (NE). Notably, the formulations exhibited considerable uptake without showing any toxicity. In conclusion, this thesis advances nanomedicine by bridging diverse applications through the exploration of NP synthesis methods. The fusion of microfluidics, DoE, and targeted formulations not only showcases a comprehensive and forward-looking approach to revolutionizing therapeutic interventions across multiple medical domains but also offers scalability. This scalability facilitates the transition of nanomedicine from bench to bedside, paving the way for efficient and personalized clinical application.Nanoparticles offer a paradigm shift in therapeutic strategies. These structures, derived from diverse materials, exhibit unique properties such as enhanced drug delivery, controlled release, and targeted therapies. Microfluidic methods, a cutting-edge technology, revolutionize the fabrication of nanoparticles by enabling precise manipulation of fluids in microchannels. This approach provides advantages like scalable production, rapid mixing, and controlled physicochemical properties. Together, nanoparticles and microfluidics redefine therapeutic approaches. For instance, the collaboration has been exemplified in the production of lipid nanoparticles encapsulating mRNA vaccines for cancer and COVID-19, showcasing a transformative leap in the field of medicine. This convergence of nanotechnology and microfluidics holds immense promise for tailored and efficient drug delivery systems, maximizing treatment efficacy while minimizing side effects. In this thesis, the result of the conducted research on nanoparticle preparation utilizing the microfluidic method and Design of Experiments (DoE) during my PhD program has been presented. Initial projects delve into the investigation of novel applications in anticancer drug delivery. Leveraging carbohydrate targeting and the unique properties of berberine, innovative liposomal formulations demonstrate enhanced anti-proliferative effects. In addressing cutaneous mycoses, the thesis introduces keratin nanoparticles as a promising antifungal drug carrier. Tannic acid (TA) crosslinking proves superior to conventional crosslinkers, ensuring stability and effectiveness in tioconazole-loaded keratin nanoparticles (TCZ-KNP). The study exemplifies a tailored approach to combat topical fungal infections. Our third study aims to assess the impact of various nanomedicines on hematological malignancies (HM), offering new possibilities for enhanced patient outcomes, where curative treatments are scarce. Conventional top-down nanomedicine development methods in preclinical research are time-consuming, motivating a bottom-up microfluidic and DoE approach to expedite production, evaluation, and prediction of a nanoparticle (NP) library composed of liposomes, lipid nanoparticles (LNP), and nanoemulsions (NE). Notably, the formulations exhibited considerable uptake without showing any toxicity. In conclusion, this thesis advances nanomedicine by bridging diverse applications through the exploration of NP synthesis methods. The fusion of microfluidics, DoE, and targeted formulations not only showcases a comprehensive and forward-looking approach to revolutionizing therapeutic interventions across multiple medical domains but also offers scalability. This scalability facilitates the transition of nanomedicine from bench to bedside, paving the way for efficient and personalized clinical applicatio

Dialectometry of Linguistic Varieties Common in the Distance between the South of Hamadan Province to the North of Khuzestan Province: Using Levenshtein Distance Approach

Dialectometry is a computational, quantitative and statistical approach, in which linguistic differences, in a selected geographical area, are examined by using specific methods and techniques. In the present study, the linguistic distances between varieties, common in the area from the south of Hamadan province to the north of Khuzestan province, and their regional distributions, have been studied by using a novel dialectometric approach. These language varieties are mostly Laki and Lori. This study is done in a library and field work method. Therefore, to do that, the distances between the equivalents of 100 words in 80 locations are measured, using Levenshtein distance which is included in RuG/L04software. After the analyzing of the linguistic distances, the outputs are presented in the forms of interpretable maps, diagrams and statistical analysis. The main results of this study are: 1. The clustering of language varieties and 2. The manner of their linguistic-geographical distribution through a linguistic continuum. Also, the study of phonetic-lexical differences between the collected linguistic data, confirms the nature of continuity of these linguistic varieties. Thus, at one end of this continuum, Laki and Lori Lorestani varieties and at the other end, Lori Bakhtiari varieties are locate Introduction Language has a continuous nature, making it difficult to establish clear-cut boundaries between different language varieties. Unlike traditional and qualitative approaches used in dialectology, which often relied on subjective methods, modern dialectometric methods should be employed due to the continuous nature of language. Hence, it is unfeasible to ascertain precise linguistic demarcations among language variations.     Quantitative methods in dialectology differ from traditional methods. Traditional approaches rely on the assumptions and linguistic features of the native speakers to determine the distribution and spread of language varieties in a particular geographic region. One of the disadvantages of these methods is the absence of consistent overlap between isoglosses and the utilization of personal preferences and opinions in choosing bundles of isoglosses. Quantitative approaches offer several advantages over traditional approaches. The advantage of using quantitative approaches compared to traditional approaches is the digital classification of data, automatic measurement of distances and frequencies, digital mapping of outputs and providing statistical analysis of linguistic data, which leads to the analysis of a large amount of linguistic data without the personal preferences of the individual researcher.     Consequently, this study involves the aggregate analysis of a vast amount of linguistic data through quantitative methods, specifically linguistic distance measurement.     The application of quantitative approaches in dialectology research, along with various dialectometric techniques and the assessment of linguistic distances in non-Iranian studies, can be traced back to the works of Seguy (1973) and Goebel (1982). Over the past ten years, dialectometry in Iran has become a focal point for numerous researchers specializing in Iranian languages and dialects. These Studies have been carried out on various common language varieties found in regions such as East Azarbaijan, West Azarbaijan, Hamadan, Mazandaran, Gorgan, Yazd, Ilam, Cherdaval and Talesh.     The preservation of a society's identity and cultural heritage is closely intertwined with the study of languages and dialects. As a result, researchers and linguists must prioritize conducting methodical studies in this field. Historically, studies on Iranian language varieties have been conducted in isolation, focusing solely on specific linguistic aspects such as phonology, morphology or syntax. Thus, numerous studies have been carried out thus far, employing both traditional and scientific methodologies, to explore different facets of language varieties (Lori and Laki) common in the examined geographical region. These investigations have primarily focused on phonology, morphology and syntax. It is important to note that these studies are solely descriptive and qualitative in nature, lacking any comparative or quantitative research elements.     Furthermore, up to now, few studies have addressed the matter of comparing different linguistic variations with a comprehensive approach, aiming to establish a systematic correlation between linguistic varieties.     Their efforts have focused on creating a linguistic atlas and developing a meticulous, scientific, and well-organized classification system for these variations. Hence, it is imperative to carry out a linguistic investigation employing a dialectometric methodology to employ contemporary analytical-computational techniques on prevalent language variations in Lorestan province and its adjacent provinces, namely Hamadan and Khuzestan. The rationale behind selecting this specific geographical scope is the extensive usage of both Lori and Laki dialects within these territories. Consequently, it holds significant value to ascertain the linguistic-geographical dispersion of these language varieties in the aforementioned regions, disregarding any geographical limitations.Methodology This study is a synchronic descriptive-analytical investigation. The RuG/L04 dialectometry and cartography software package was employed to conduct this study. Initially, 100 lexical entries were gathered from 80 different locations. The research database was sourced from three national dialectology projects in Lorestan and Khuzestan provinces, as well as from the field research conducted by the researchers. The participants in this study encompass both males and females, ranging in age from 20 to 70 years, with an average educational attainment of a high school diploma. After transcribing 8000 lexical forms, the geographical coordinates for each location were determined by utilizing Google Earth software.     Subsequently, in order to calculate the linguistic distance index, Levenshtein distance algorithm was applied to the data as one of the aggregate analysis approaches. The resulting distance, obtained from the 80x80 matrix, represents a quantitative index within the range of natural numbers. In the subsequent phase of the study, diverse subprograms were employed to categorize the acquired language types. These categorizations are then presented in the form of diagrams, tables, and maps.Results Upon analyzing the acquired outcomes, it was discovered that the linguistic variations being investigated form a continuous language continuum, devoid of any distinct boundaries (in contrast to traditional dialectology approaches). This continuum commences from the Laki and Lori varieties of Lorestan in the southern region of Hamadan province, extending all the way to the Bakhtiari varieties in the northern part of Khuzestan province. The attribute of continuity is also evident in the phonetic differences and alternations observed among the three primary language varieties, namely Laki, Lori Bakhtiari, and Lori Lorestani.Conclusion The greater language distance and difference between Laki and Lori varieties (as indicated in equation 2.5) confirms the belonging of each of these varieties to a different language family; Also, due to belonging to a common language family (Southwestern Iranian), there is less linguistic distance (equal to 1.5) and more linguistic similarity between two varieties of Lori. The obtained Pearson correlation coefficient between the linguistic varieties under study is r=0.88, indicating a strong and statistically significant correlation percentage. This validates the findings of the research and highlights the effectiveness of utilizing Levenshtein's distance dialectometric approach in identifying the primary linguistic clusters of the examined varieties and confirming the continuity nature of the language

3D-printed EVA-based patches manufactured by direct powder extrusion for personalized transdermal therapies

In recent years, 3D printing has attracted great interest in the pharmaceutical field as a promising tool for the on-demand manufacturing of patient-centered pharmaceutical forms. Among the existing 3D printing techniques, direct powder extrusion (DPE) resulted as the most practical approach thanks to the possibility to directly process excipients and drugs in a single step. The main goal of this work was to determine whether different grades of ethylene vinyl acetate (EVA) copolymer might be employed as new feedstock materials for the DPE technique to manufacture transdermal patches. By selecting two model drugs with different thermal behavior, (i.e., ibuprofen and diclofenac sodium) we also wanted to pay attention to the versatility of EVA excipient in preparing patches for customized transdermal therapies. EVA was combined with 30% (w/w) of each model drugs. The physicochemical composition of the printed devices was investigated through Fourier-transform infrared spectroscopy, differential scanning calorimetry, and thermogravimetric analyses. FT-IR spectra confirmed that the starting materials were effectively incorporated into the final formulation, and thermal analyses demonstrated that the extrusion process altered the crystalline morphology of the raw polymers inducing the formation of crystals at lower thicknesses. Lastly, the drug release and permeation profile of the printed systems was evaluated for 48 hours and showed to be dependent on the VA content of the EVA grade (74.5% of ibuprofen released from EVA 4030AC matrix and 12.6% of diclofenac sodium released from EVA1821A matrix). Hence, this study demonstrated that EVA and direct powder extrusion technique could be promising tools for manufacturing transdermal patches. By selecting the EVA grade with the appropriate VA content, drugs with dissimilar melting points could be printed preserving their thermal stability. Moreover, the desired drug release and permeation profile of the drug can be achieved, representing an important advantage in terms of personalized medicine

Cell-imprinted substrates: in search of nanotopographical fingerprints that guide stem cell differentiation

Cell-imprinted substrates direct stem cell differentiation into various lineages, suggesting the idea of lineage-specific nanotopography. We herein examined the surface topography of five different imprinted cell patterns using AFM imaging and statistical analysis of amplitude, spatial, and hybrid roughness parameters. The results suggest that different cell imprints possess distinguished nanotopographical features

A Hydrogen-Bonded Extracellular Matrix-Mimicking Bactericidal Hydrogel with Radical Scavenging and Hemostatic Function for pH-Responsive Wound Healing Acceleration

Generation of reactive oxygen species, delayed blood clotting, prolonged inflammation, bacterial infection, and slow cell proliferation are the main challenges of effective wound repair. Herein, a multifunctional extracellular matrix‐mimicking hydrogel is fabricated through abundant hydrogen bonding among the functional groups of gelatin and tannic acid (TA) as a green chemistry approach. The hydrogel shows adjustable physicochemical properties by altering the concentration of TA and it represents high safety features both in vitro and in vivo on fibroblasts, red blood cells, and mice organs. In addition to the merit of facile encapsulation of cell proliferation‐inducing hydrophilic drugs, accelerated healing of skin injury is obtained through pH‐dependent release of TA and its multifaceted mechanisms as an antibacterial, antioxidant, hemostatic, and anti‐inflammatory moiety. The developed gelatin‐TA (GelTA) hydrogel also shows an outstanding effect on the formation of extracellular matrix and wound closure in vivo via offered cell adhesion sites in the backbone of gelatin that provide increased re‐epithelialization and better collagen deposition. These results suggest that the multifunctional GelTA hydrogel is a promising candidate for the clinical treatment of full‐thickness wounds and further development of wound dressing materials that releases active agents in the neutral or slightly basic environment of infected nonhealing wounds.Peer reviewe

A microfluidic approach to fabricate sucrose decorated liposomes with increased uptake in breast cancer cells

Developing targeted drug delivery systems is an urgent need to decrease the side effects and increase the drug's efficiency. Most cancer cells show an increased sugar consumption compared to healthy cells due to the deregulation of sugar transporters. Consequently, liposomes, as a biocompatible nanocarrier, could be surface decorated by sugars to enhance drug targeting into cancer cells. Our work outlines a new strategy to easily manufacture sucrose decorated liposomes using sucrose stearate, a biocompatible and biodegradable non-ionic surfactant, with a scalable microfluidic approach. Sucrose decorated liposomes were loaded with berberine hydrochloride, a well-known phytochemical compound to investigate its effects on triple-negative breast cancer cells (MDA-MB-231). Using the microfluidic manufacturing system, we prepared berberine-loaded liposomes using a mixture of phosphatidylcholine and cholesterol with and without sucrose stearate with a size up to 140 nm and narrow polydispersity. Stability was confirmed for 90 days, and the in vitro release profile was evaluated. The formulations showed acceptable in vitro biocompatibility and significantly higher anti-proliferative effect on MDA-MB-231 cell line. These results have been confirmed by an increased uptake evaluated by flow cytometry and confocal microscopy. Taken together, our findings represent an innovative, easy, and scalable approach to obtain sugar decorated liposomal formulations without any surface-chemistry reactions. They can be potentially used as an anticancer targeted drug delivery system

Engineered substrates incapable of induction of chondrogenic differentiation compared to the chondrocyte imprinted substrates

It is well established that surface topography can affect cell functions. However, finding a reproducible and reliable method for regulating stem cell behavior is still under investigation. It has been shown that cell imprinted substrates contain micro- and nanoscale structures of the cell membrane that serve as hierarchical substrates, can successfully alter stem cell fate. This study investigated the effect of the overall cell shape by fabricating silicon wafers containing pit structure in the average size of spherical-like chondrocytes using photolithography technique. We also used chondrocyte cell line (C28/I2) with spindle-like shape to produce cell imprinted substrates. The effect of all substrates on the differentiation of adipose-derived mesenchymal stem cells (ADSCs) has been studied. The AFM and scanning electron microscopy images of the prepared substrates demonstrated that the desired shapes were successfully transferred to the substrates. Differentiation of ADSCs was investigated by immunostaining for mature chondrocyte marker, collagen II, and gene expression of collagen II, Sox9, and aggrecan markers. C28/I2 imprinted substrate could effectively enhanced chondrogenic differentiation compared to regular pit patterns on the wafer. It can be concluded that cell imprinted substrates can induce differentiation signals better than engineered lithographic substrates. The nanostructures on the cell-imprinted patterns play a crucial role in harnessing cell fate. Therefore, the patterns must include the nano-topographies to have reliable and reproducible engineered substrates.LMIS

Microfluidic manufacturing of tioconazole loaded keratin nanocarriers: Development and optimization by design of experiments

: Fungal infections of the skin, nails, and hair are a common health concern affecting a significant proportion of the population worldwide. The current treatment options include topical and systematic agents which have low permeability and prolonged treatment period, respectively. Consequently, there is a growing need for a permeable, effective, and safe treatment. Keratin nanoparticles are a promising nanoformulation that can improve antifungal agent penetration, providing sustainable targeted drug delivery. In this study, keratin nanoparticles were prepared using a custom-made 3D-printed microfluidic chip and the manufacturing process was optimized using the design of experiments (DoE) approach. The total flow rate (TFR), flow rate ratio (FRR), and keratin concentration were found to be the most influential factors of the size and polydispersity index (PDI) of the nanoparticles. The crosslinking procedure by means of tannic acid as safe and biocompatible compound was also optimized. Keratin nanoparticles loaded with a different amount of tioconazole showed a size lower than 200 nm, a PDI lower than 0.2 and an encapsulation efficiency of 91 ± 1.9 %. Due to their sustained drug release, the formulations showed acceptable in vitro biocompatibility. Furthermore, a significant inhibitory effect compared to the free drug against Microsporum canis