Drug-loaded erythrocytes: Modern approaches for advanced drug delivery for clinical use.

Scientific organizations worldwide are striving to create drug delivery systems that provide a high local concentration of a drug in pathological tissue without side effects on healthy organs in the body. Important physiological properties of red blood cells (RBCs), such as frequent renewal ability, good oxygen carrying ability, unique shape and membrane flexibility, allow them to be used as natural carriers of drugs in the body. Erythrocyte carriers derived from autologous blood are even more promising drug delivery systems due to their immunogenic compatibility, safety, natural uniqueness, simple preparation, biodegradability and convenience of use in clinical practice. This review is focused on the achievements in the clinical application of targeted drug delivery systems based on osmotic methods of loading RBCs, with an emphasis on advancements in their industrial production. This article describes the basic methods used for encapsulating drugs into erythrocytes, key strategic approaches to the clinical use of drug-loaded erythrocytes obtained by hypotonic hemolysis. Moreover, clinical trials of erythrocyte carriers for the targeted delivery are discussed. This article explores the recent advancements and engineering approaches employed in the encapsulation of erythrocytes through hypotonic hemolysis methods, as well as the most promising inventions in this field. There is currently a shortage of reviews focused on the automation of drug loading into RBCs; therefore, our work fills this gap. Finally, further prospects for the development of engineering and technological solutions for the automatic production of drug-loaded RBCs were studied. Automated devices have the potential to provide the widespread production of RBC-encapsulated therapeutic drugs and optimize the process of targeted drug delivery in the body. Furthermore, they can expedite the widespread introduction of this innovative treatment method into clinical practice, thereby significantly expanding the effectiveness of treatment in both surgery and all areas of medicine.

Risk Factors for Primary Pulmonary TB in Almaty Region, Kazakhstan: A Matched Case-Control Study.

BACKGROUND: This study examined the association between incident pulmonary tuberculosis (TB) and social and behavioral characteristics in Almaty Oblast, Kazakhstan from 2012 to 2013. METHODS: We used a matched case-control design to estimate the role of factors for acquiring pulmonary TB. Totally 324 individuals were recruited from Sep 2012 to Mar 2013. Participants included 110 TB index cases with newly detected pulmonary TB. Each case was matched with one household and one community control. A total of 107 household and 107 community controls were included to the study. Adjusted odds ratios measuring associations between TB and risk factors were calculated by using a conditional multiple logistic regression analysis. RESULTS: TB cases were more likely to be younger, recent smokers and have diabetes, when compared to household controls. Between TB cases and community controls, TB was significantly associated with age, non-married family status, living in a rented home, recent smoker, and having diabetes. Comparing TB cases with community controls, we found that foreign birth was marginally associated with incident TB case status. CONCLUSION: Our findings confirm the role of modifiable risk factors for TB in Kazakhstan; highlighting the importance of developing interventions addressing social determinants and proximate risk factors for high TB burden regions.

Red blood cell ghosts as promising drug carriers to target wound infections.

Autologous red blood cell ghosts (RBC ghosts) can carry cytokines to the sites of inflammation. The targeting moiety of the RBC ghosts is associated with the nature of purulent inflammation, where the erythrocytes are phagocyted and encapsulated drugs are released. In the present study we have investigated the healing potential of RBC ghosts loaded with cytokine IL-1ß and antibiotic. Additionally, the pharmacokinetic properties of RBC ghosts loaded with IL-1ß were studied. 35 Male Wistar rats (250-300g) were used in the pharmacokinetic study and in a wound infection model where a suspension of Staphylococcus aureus was placed into a surgical cut of the skin and subcutaneous tissue in the femoral region. In order to monitor progression of the wound repair processes, wound swabs or aspiration biopsies were taken for analyses on the 1st-6th days. Wound repair dynamics assessment was based on suppression of S. aureus growth, signs of pain, time of disappearance of pus and infiltration around the wound. Visual observations, as well as microbiological and cytological analysis of wound exudates demonstrated a significant acceleration of healing processes in a group of animals treated with a local injection of IL-1ß and ceftriaxone encapsulated into RBC ghosts when compared to the animals treated either with a local or IM injection of free drugs. For the pharmacokinetic study, single IV injections of either free or encapsulated IL-1ß were made and the concentration of IL-1ß in serum samples and tissue homogenates were determined. Encapsulation in RBC ghosts improved pharmacokinetic profiles of IL-1ß by increasing the half-life, reducing its clearance, and increasing the deposition of the drug in the liver, spleen and lungs. These data suggest that RBC ghosts are effective drug carriers for targeted delivery of cytokines to the sites of inflammation, and have a potential for improving the treatment outcomes of purulent diseases.

Proteomic analysis of cellular response induced by boron neutron capture reaction in human squamous cell carcinoma SAS cells.

To understand the mechanism of cell death induced by boron neutron capture reaction (BNCR), we performed proteome analyses of human squamous tumor SAS cells after BNCR. Cells were irradiated with thermal neutron beam at KUR after incubation under boronophenylalanine (BPA)(+) and BPA(-) conditions. BNCR mainly induced typical apoptosis in SAS cells 24h post-irradiation. Proteomic analysis in SAS cells suggested that proteins functioning in endoplasmic reticulum, DNA repair, and RNA processing showed dynamic changes at early phase after BNCR and could be involved in the regulation of cellular response to BNCR. We found that the BNCR induces fragments of endoplasmic reticulum-localized lymphoid-restricted protein (LRMP). The fragmentation of LRMP was also observed in the rat tumor graft model 20 hours after BNCT treatment carried out at the National Nuclear Center of the Republic of Kazakhstan. These data suggest that dynamic changes of LRMP could be involved during cellular response to BNCR.

Histological and biochemical analysis of DNA damage after BNCT in rat model.

To understand the mechanism of tumor cell death induced by boron neutron capture therapy (BNCT) and to optimize BNCT condition, we used rat tumor graft models and histological and biochemical analyses were carried out focusing on DNA damage response. Rat lymphosarcoma cells were grafted subcutaneously into male Wister rats. The rats with developed tumors were then treated with neutron beam irradiation 45min after injection of 330mg/kg bodyweight boronophenylalanine ((10)BPA) (+BPA) or saline control (-BPA). BNCT was carried out in the National Nuclear Center of the Republic of Kazakhstan (neutron flux: 1×10(9)nvt/s, fluence: 6×10(11)nvt) with the presence of background γ-irradiation of 33Gy. 6 and 20h after BNCT treatment, tumors were resected, fixed and subjected to immunohistochemistry and biochemical analyses. Immunostaining of nuclei showed that double strand break (DSB) marker gamma H2AX staining was high in 20h/+BPA sample but not in 20h/-BPA samples. Poly(ADP-ribose), DSB and single strand break markers of DNA, also demonstrated this tendency. These two markers were observed at low levels in unirradiated tissues or 6h after BNCT either under -BPA and +BPA conditions. HMGB1 level increased in 6h/+BPA but not in 6h/-BPA or 20h/+BPA samples. The persistent staining of γH2AX and poly(ADP-ribose) in +BPA group suggests accumulated DSB damage after BNCT. The early HMGB1 upregulation and γH2AX and poly(ADP-ribose) observed later might be the markers for monitoring the DNA damage induced by BNCT.

Pharmacokinetic Properties of Cytokines in Their Targeted Delivery Based on Autologous Erythrocyte Pharmacocytes.

INTRODUCTION: Using autologous erythrocytes as drug carriers for targeted delivery of cytokines to the sites of inflammation could potentially provide new opportunities for treatment of patients with purulent diseases. The targeted characteristic of erythrocytes is associated with the nature of purulent inflammation, where a large amount of erythrocytes is phagocytized and drugs encapsulated into the erythrocytes could be easily released. On the other hand, autologous erythrocytes meet all the criteria for the ideal drug carrier. They are nontoxic, not immunogenic, and able to bear a large number of drug molecules while preserving an original conformation of the drugs. Thus, in this study, we aimed to analyze pharmacokinetic profiles of IL-1ß encapsulated into erythrocytes' ghosts (pharmacocytes) in comparison to intravenously injected free IL-1ß. MATERIAL AND METHODS: Albino rats were randomly divided into two groups, each group receiving a different kind of IV injection via the tail vein. Group A (control) received 500 µg of free IL-1ß, and group B received an injection of 1 ml of pharmacocytes loaded with 500 µg of test substance. At fixed time points after injection (15, 30, 60, 180, 540, 720, and 1,440 minutes) serum samples were collected. Homogenates of liver, spleen, lung, heart, kidney, and adipose tissue were obtained 24 hours after injections. Concentration of the tested substance in the collected organs and blood plasma were measured by ELISA. RESULTS: We have observed an increased half-life period (T1/2) for encapsulated IL-1ß compared to the control. T1/2 for free IL-1ß was one hour, while administration of loaded pharmacocytes allowed the half-life period to increase by more than 15 fold (1,043.40 ± 137.92 min) preserving high level of IL-1ß activity in the blood samples up to 24 hours. The increased time of IL-1ß presence in the body when administered in the form of pharmacocytes could be explained by reduction of elimination constant (Cel) by 1.6 fold, and clearance (CLel) by more than 100 fold. We also observed an increased concentration of IL-1ß in liver, spleen, and lung over at least 24 hours. When administered in free form, IL-1ß disappeared from these organs within 6 hours. CONCLUSIONS: Pharmacocytes have shown to improve pharmacokinetic profiles of IL-1ß by increasing the half-life period of the cytokine, reducing its clearance and elimination as well as increasing the deposition of the drug in liver, spleen and lungs. These data suggest that pharmacocytes be effective drug carriers for targeted delivery of cytokines to the sites of inflammation and have a potential for improving the treatment outcomes of purulent diseases.

Vitamin D Receptor Gene Polymorphisms in Susceptibility to Tuberculosis in the Kazakh Population in Almaty and Almaty Area.

INTRODUCTION: Vitamin D receptor (VDR) plays an important role in activating the immune response against various infectious agents. It is known that the active metabolite of ligand receptor Vitamin D (1,25 - dihydroxyvitamin D) is encoded by VDR and helps mononuclear phagocytes to suppress the intracellular growth of M. tuberculosis. The VDR gene harbors approximately 200 polymorphisms, some of which are linked to differences in receptor Vitamin D uptake and therefore can be considered as candidate disease risk variants. The relation between VDR gene polymorphisms and susceptibility to TB has been studied in different populations. There is not a great deal of information regarding the association of these SNPs with TB risk in the Kazakh population. The four most commonly investigated VDR polymorphisms in association with different diseases, including susceptibility to tuberculosis, are located in exon 2 (rs2228570 or FokI), intron 8 (rs1544410 or BsmI and rs7975232 or ApaI), and exon 9 (rs731236 or TaqI). The aim of our study was to determine whether these four VDR gene single nucleotide polymorphisms were associated with TB and whether they were a risk for the development of TB in the Kazakh Population in Almaty city and Almaty area. METHODS: This study was a hospital-based case-control analysis of 283 individuals (99 TB patients and 184 healthy controls). Genotyping was performed by Taqman SNP allelic discrimination using commercial TaqMan SNP Genotyping assays. Statistical analysis was conducted using SPSS Version 19.0 software. RESULTS: Genotype frequencies for the Kazakh population are close to world (HapMap) data on Asian populations. FokI and ApaI polymorphisms genotypes tend to be associated with TB risk under the co-dominant model [OR=1.18; 95%CI: (0.68, 2.07), p=0.15] for FokI and [OR=1.33; 95%CI: (0.61, 2.91), p=0.6] for ApaI. No significant association between the disease and TaqI, BsmI genotypes was observed. CONCLUSIONS: In summary, we explored potential associations between SNPs in the VDR (FokI, ApaI) gene and susceptibility to tuberculosis in the Kazakh Population, which requires further detailed analysis with a larger sample size and greater geographic diversity including other regions of Kazakhstan.

Biopharmaceutical characteristics of autologous red blood cells ghosts containing cytokines and antibiotics.

INTRODUCTION: Transport systems based on autologous red blood cells for targeted drug delivery can be considered as a promising approach in the treatment of surgical infections. Experimental studies have revealed the feasibility of targeted drug delivery by encapsulation of cytokines and antibiotics into autologous erythrocyte ghosts. PURPOSE: To study biopharmaceutical characteristics of autologous erythrocyte ghosts containing cytokines and antibiotics (pharmacocytes). MATERIAL AND METHODS: The erythrocyte pharmacocytes were prepared by the hypotonic hemolysis method, or the use of human red blood cells. The association and dissociation indicators of rifampicin and cytokine substances with the erythrocyte ghosts were conducted using standard methods. RESULTS: We have defined the following extracellular concentrations to be optimal for deposition of drug substances into pharmacocytes: for rifampicin - 10 000 µ/ml, erythropoietin - 1000 IU/ml, TNF-a - 5000 IU/ml, IL-1-ß - 5000 U/ml, IFN-γ - 10 000ME/ml, IL-2 - 50 000 IU/ml, angiogenin - 0.04 mg/ml. Two types of correlations of cytokines and pharmacocytes were identified. In this study, we found that 40-60 % of the erythropoietin, IFN- γ and angiogenin were bound to red blood cells ghosts, more than 10% of which were bound irreversibly. For TNF-a, IL-1-ß and IL-2, the red blood cells ghosts were capable of binding and depositing within 10-20 % of the input extracellular concentration, and these bindings were almost completely reversible. The rifampicin was bound by red blood cells ghosts with 5 % efficiency and also completely reversibly. CONCLUSION: The study has shown the effectiveness of inclusion of the studied components, such as erythropoietin, IFN-γ and angiogenin into the red blood cells ghosts, with significant efficiency (40-60 %). It presents the potential of using this system in targeted delivery of cytokines and antibiotics for treatment of surgical infections, thus facilitating the reduction in toxicity and adverse systemic effects of drugs and improving the treatment results.