Preparation, Characterization, and Radiolabeling of Anti-HER2 scFv With Technetium Tricarbonyl and Stability Studies.

Breast cancer is the most common diagnosed cancer, and the second cause of cancer death among women, worldwide. HER2 overexpression occurred in approximately 15% to 20% of breast cancers. Invasive biopsy method has been used for detection of HER2 overexpression. HER2-targeted imaging via an appropriate radionuclide is a promising method for sensitive and accurate identification of HER2+ primary and metastatic lesions. 99mTc-anti-HER2 scFv can specifically target malignancies and be used for diagnosis of the cancer type and metastasis as well as treatment of breast cancer. We radiolabeled anti-HER2 scFv that was expressed in Escherichia coli and purified through Ni-NTA resin under native condition with 99mTc-tricarbonyl formed from boranocarbonate. HER2-based ELISA, BCA, TLC, and HPLC were used in this study. In the current study, anti-HER2 scFv was lyophilized before radiolabeling. It was found that freeze-drying did not change the binding activity of anti-HER2 scFv to HER2. Results demonstrated direct anti-HER2 scFv radiolabeling by 99mTc-tricarbonyl to hexahistidine sequence (His-tag) without any changes in biological activity and radiochemical purity of around 98%. Stability analysis revealed that 99mTc-anti-HER2 scFv is stable for at least 24 h in PBS buffer, normal saline, human plasma proteins, and histidine solution.

Evaluating the efficacy of immobilized metal affinity chromatography (IMAC) for host cell protein (HCP) removal from anti-HER2 scFv expressed in Escherichia coli.

Host cell proteins (HCPs) are process-related impurities that have influence on product safety and efficacy. HCPs should effectively be removed by chromatographic steps in downstream purification process. In this study, we aimed to evaluate the efficacy of immobilized-metal affinity chromatography (IMAC) for separation of HCPs from anti-HER2 single chain fragment variable (scFv) expressed in E. coli. This study explored how different purification conditions including native, denaturing and hybrid affect HCP level in purified anti-HER2 scFv. Furthermore, the effects of NaCl concentration in wash buffer as well as imidazole concentration in wash and elution buffer on purification yield and HCP level in purified anti-HER2 scFv were evaluated. It was found that increasing imidazole concentration in wash and elution buffers in native conditions reduced the yield of anti-HER2 scFv purification. However, enhancing NaCl concentration in wash buffer in purification under native conditions led to significant increase in the amount of anti-HER2 scFv without any change in protein purity. Herein, none of the IMAC purification methods conducted on soluble cytoplasmic proteins under native conditions could reduce the amount of HCP to acceptable level. HCP content was only lowered to ˂ 10 ppm when inclusion bodies were purified under hybrid conditions. Furthermore, increasing imidazole concentration in wash buffer in purification under hybrid conditions led to significant increase in eluted anti-HER2 scFv concentration, while HCP content was also increased in this condition. Overall, purification under hybrid conditions using wash buffer containing 40 mM imidazole resulted in the highest yield and acceptable level of HCP.

Synthesis of 4,6-diphenylpyrimidin-2-ol derivatives as new benzodiazepine receptor ligands.

Benzodiazepines (BZDs) have been widely used in neurological disorders such as insomnia, anxiety, and epilepsy. The use of classical BZDs, e.g., diazepam, has been limited due to adverse effects such as interaction with alcohol, ataxia, amnesia, psychological and physical dependence, and tolerance. In the quest for new benzodiazepine agonists with more selectivity and low adverse effects, novel derivatives of 4,6-diphenylpyrimidin-2-ol were designed, synthesized, and evaluated. In this series, compound 2, 4-(2-(benzyloxy)phenyl)-6-(4-fluorophenyl)pyrimidin-2-ol, was the most potent analogue in radioligand binding assay with an IC50 value of 19 nM compared to zolpidem (IC50 = 48 nM), a nonbenzodiazepine central BZD receptor (CBR) agonist. Some compounds with a variety of affinities in radioligand receptor binding assay were selected for in vivo evaluations. Compound 3 (IC50 = 25 nM), which possessed chlorine instead of fluorine in position 4 of the phenyl ring, exhibited an excellent ED50 value in most in vivo tests. Proper sedative-hypnotic effects, potent anticonvulsant activity, appropriate antianxiety effect, and no memory impairment probably served compound 3, a desirable candidate as a benzodiazepine agonist. The pharmacological effects of compound 3 were antagonized by flumazenil, a selective BZD receptor antagonist, confirming the BZD receptors' involvement in the biological effects of the novel ligand.

5-[Aryloxypyridyl (or nitrophenyl)]-4H-1,2,4-triazoles as novel flexible benzodiazepine analogues: Synthesis, receptor binding affinity and lipophilicity-dependent anti-seizure onset of action.

A new series of 5-(2-aryloxy-4-nitrophenyl)-4H-1,2,4-triazoles and 5-(2-aryloxy-3-pyridyl)-4H-1,2,4-triazoles, possessing C-3 thio or alkylthio substituents, was synthesized and evaluated for their benzodiazepine receptor affinity and anti-seizure activity. These analogues revealed similar to significantly superior affinity to GABAA/benzodiazepine receptor complex (IC50 values of 0.04-4.1 nM), relative to diazepam as the reference drug (IC50 value of 2.4 nM). To determine the onset of anti-seizure activity, the time-dependent effectiveness of i.p. administration of compounds on pentylenetetrazole induced seizure threshold was studied and a very good relationship was observed between the lipophilicity (cLogP) and onset of action of studied analogues (r2 = 0.964). The minimum effective dose of the compounds, determined at the time the analogues showed their highest activity, was demonstrated to be 0.025-0.1 mg/kg, relative to diazepam (0.025 mg/kg).

Design, synthesis, radiolabeling and biological evaluation of new urea-based peptides targeting prostate specific membrane antigen.

Early diagnosis of Prostate cancer (PCa) plays a vital role in successful treatment increasing the survival rate of patients. Prostate Specific Membrane Antigen (PSMA) is over-expressed in almost all types of PCa. The goal of present study is to introduce new 99mTc-labeled peptides as a PSMA inhibitor for specific detection of PCa at early stages. Based on published PSMA-targeting compounds, a set of peptides bearing the well-known Glu-Urea-Lys pharmacophore and new non-urea containing pharmacophore were designed and assessed by in silico docking studies. The selected peptides were synthesized and radiolabeled with 99mTc. The in-vitro tests (log P, stability in normal saline and fresh human plasma, and affinity toward PSMA-positive LNCaP cell line) and in-vivo characterizations of radiopeptides (biodistribution and Single Photon Emission Computed Tomography-Computed Tomography (SPECT-CT) imaging in normal and tumour-bearing mice) were performed. The peptides 1-3 containing Glu-Urea-Lys and Glu-GABA-Asp as pharmacophores were efficiently interacted with crystal structure of PSMA and showed the highest binding energies range from -8 to -11.2 kcal/mol. Regarding the saturation binding test, 99mTc-labeled peptide 1 had the highest binding affinity (Kd = 13.58 nM) to PSMA-positive cells. SPECT-CT imaging and biodistribution studies showed high kidneys and tumour uptake 1 h post-injection of radiopeptide 1 and 2 (%ID/g tumour = 3.62 ± 0.78 and 1.8 ± 0.32, respectively). 99mTc-peptide 1 (Glu-urea-Lys-Gly-Ala-Asp-Naphthylalanine-HYNIC-99mTc) exhibited the highest binding affinity, high radiochemical purity, the most stability and high specific accumulation in prostate tumour lesions. 99mTc-peptide 1 being of comparable efficacy and pharmacokinetic properties with the well-known PET tracer (68Ga-PSMA-11) seems to be applied as a promising SPECT imaging agent to early diagnose of PCa and consequently increase survival rate of patients.

Synthesis and radioligand-binding assay of 2,5-disubstituted thiadiazoles and evaluation of their anticonvulsant activities.

In this study, a number of 2,5-disubstituted 1,3,4-thiadiazoles were synthesized using an appropriate synthetic route, and their anticonvulsant activity was determined by the maximal electroshock seizure (MES) test and their neurotoxicity was evaluated by the rotarod test. Additionally, their hypnotic activity was tested using the pentobarbital-induced sleep test. Compounds 7 (ED50 = 1.14 and 2.72 µmol/kg in the MES and sleep tests, respectively) and 11 (ED50 = 0.65 and 2.70 µmol/kg in the MES and sleep tests, respectively) were the most potent ones in the sleep test and anticonvulsant test, showing a comparable activity with diazepam as the reference drug. The results of in vivo studies, especially the antagonistic effects of flumazenil, and also the radioligand-binding assay confirmed the involvement of benzodiazepine (BZD) receptors in the anticonvulsant and hypnotic activity of compounds 7 and 11. Finally, the docking study of compound 11 in the BZD-binding site of the GABAA (gamma-aminobutyric acid) receptor confirmed the possible binding of the compound to the BZD receptors. We concluded that the novel 1,3,4-thiadiazole derivatives with appropriate substitution at positions 2 and 5 of the heterocyclic ring had a good affinity to BZD receptors and showed significant efficacy in the pharmacological tests.

Initial In Vitro and In Vivo Evaluation of a Novel CCK2R Targeting Peptide Analog Labeled with Lutetium-177.

Targeting of cholecystokinin-2 receptor (CCK2R) expressing tumors using radiolabeled minigastrin (MG) analogs is hampered by rapid digestion of the linear peptide in vivo. In this study, a new MG analog stabilized against enzymatic degradation was investigated in preclinical studies to characterize the metabolites formed in vivo. The new MG analog DOTA-DGlu-Pro-Tyr-Gly-Trp-(N-Me)Nle-Asp-1Nal-NH2 comprising site-specific amino acid substitutions in position 2, 6 and 8 and different possible metabolites thereof were synthesized. The receptor interaction of the peptide and selected metabolites was evaluated in a CCK2R-expressing cell line. The enzymatic stability of the 177Lu-labeled peptide analog was evaluated in vitro in different media as well as in BALB/c mice up to 1 h after injection and the metabolites were identified based on radio-HPLC analysis. The new radiopeptide showed a highly increased stability in vivo with >56% intact radiopeptide in the blood of BALB/c mice 1 h after injection. High CCK2R affinity and cell uptake was confirmed only for the intact peptide, whereas enzymatic cleavage within the receptor specific C-terminal amino acid sequence resulted in complete loss of affinity and cell uptake. A favorable biodistribution profile was observed in BALB/c mice with low background activity, preferential renal excretion and prolonged uptake in CCK2R-expressing tissues. The novel stabilized MG analog shows high potential for diagnostic and therapeutic use. The radiometabolites characterized give new insights into the enzymatic degradation in vivo.

Labrasol-Enriched Nanoliposomal Formulation: Novel Approach to Improve Oral Absorption of Water-Insoluble Drug, Carvedilol.

The purpose of the current study was to develop a novel liposomal formulation to improve the oral bioavailability of carvedilol, a Biopharmaceutics Classification System class II with poor aqueous solubility and extensive presystemic metabolism. Conventional and various surfactant-enriched carvedilol-loaded liposomes were prepared by thin film hydration technique and physicochemical properties of liposomes (including size, encapsulation efficiency, release behavior, and morphology) were evaluated. To assess the oral bioavailability, in vivo studies were carried out in eight groups of male Wistar rats (n = 6) and the drug plasma concentration was determined. Conventional and surfactant containing liposomes showed average particle size of 76-104 nm with a narrow size distribution, high encapsulation efficiency (80%≤) and a sustained release profile in simulated intestinal fluid. Compared to the suspension, conventional and Labrasol containing liposomes significantly improved the oral bioavailability and peak plasma concentration of carvedilol. Biocompatibility studies (cell cytotoxicity and histopathological analyses) showed that the enhancing effect might be achieved without any apparent toxicity in the intestine. Decreased oral absorption of carvedilol nanovesicles by using a chylomicron flow blocker indicated contribution of lymphatic transport in nanocapsules absorption. The results reported the successful development of biocompatible Labrasol-enriched carvedilol nanoliposomal formulation with a significant oral enhancement capability. Graphical Abstract ᅟ.

Radiolabeling of Preformed Niosomes with [99mTc]: In Vitro Stability, Biodistribution, and In Vivo Performance.

Nanocarriers radiolabeled with [99mTc] can be used for diagnostic imaging and radionuclide therapy, as well as tracking their pharmacokinetic and biodistribution characteristics. Due to the advantages of niosomes as an ideal drug delivery system, in this study, the radiolabeling procedure of niosomes by [99mTc]-HMPAO complexes was investigated and optimized. Glutathione (GSH)-loaded niosomes were prepared using a thin-film hydration method. To label the niosomes with [99mTc], the preformed GSH-loaded niosomes were incubated with the [99mTc]-HMPAO complex and were characterized for particle size, size distribution, zeta potential, morphology, and radiolabeling efficiency (RE). The effects of GSH concentration, incubation time, incubation temperature, and niosomal composition on RE were investigated. The biodistribution profile and in vivo SPECT/CT imaging of the niosomes and free [99mTc]-HMPAO were also studied. Based on the results, all vesicles had nano-sized structure (160-235 nm) and negative surface charge. Among the different experimental conditions that were tested, including various incubation times, incubation temperatures, and GSH concentrations, the optimum condition that resulted in a RE of 92% was 200-mM GSH and 15-min incubation at 40°C. The in vitro release study in plasma showed that about 20% of radioactivity was released after 24 h, indicating an acceptable radiolabeling stability in plasma. The biodistribution of niosomes was clearly different from the free radiolabel. Niosomes carrying radionuclide were successfully used for tracking the in vivo disposition of these carriers and SPECT/CT imaging in rats. Furthermore, biodistribution studies in tumor-bearing mice revealed higher tumor accumulation of the niosomal formulation as compared with [99mTc]-HMPAO.

Novel agonists of benzodiazepine receptors: design, synthesis, binding assay and pharmacological evaluation of 1,2,4-triazolo[1,5-a]pyrimidinone and 3-amino-1,2,4-triazole derivatives.

Agonists of benzodiazepine (BZD) binding site in GABA receptors are widely used in clinical practice. In spite of their benefits they have several side effects, so synthesis of new agonists of these receptors to get more specific effect and better profile of adverse drug reactions is still continued. Novel BZD agonists were designed based on the pharmacophore/receptor model of BZD binding site of GABAA receptor. Energy minima conformers of the designed compounds and estazolam, a known BZD receptor agonist, were well superimposed in conformational analysis. Docking studies revealed that the carbonyl group of the compound 4c, 3-(2-chlorobenzyl)-5-methyl-2-phenyl-[1,2,4]triazolo[1,5-a]pyrimidin-7(3H)-one, was near the nitrogen moiety of triazole ring of estazolam providing the hydrogen bond acceptor in proper direction in the BDZ-binding site of GABAA receptor model (α1ß2ϒ2). The designed compounds were synthesized and their in vitro affinity for the central BZD receptor was determined. Most of the novel compounds had better affinity for the BZD site of action on GABAA receptor complex than diazepam. Finally, the novel compound 4c with the best affinity in radioligand receptor binding assay (Ki=0.42 nM and IC50=0.68 nM) was selected as candidate for in vivo evaluation. This compound showed significant hypnotic activity and weak anticonvulsant effect with no impairment on learning and memory performance in mouse. The pharmacological effects of the compound 4c were antagonized by flumazenil, a BZD antagonist, which confirms the involvement of BZD receptors in the biological effects of the novel ligand.

Synthesis, receptor affinity and effect on pentylenetetrazole-induced seizure threshold of novel benzodiazepine analogues: 3-Substituted 5-(2-phenoxybenzyl)-4H-1,2,4-triazoles and 2-amino-5-(phenoxybenzyl)-1,3,4-oxadiazoles.

The new series of 5-(2-phenoxybenzyl)-4H-1,2,4-triazoles, possessing C-3 thio, alkylthio and ethoxy substituents, and 2-amino-5-(2-phenoxybenzyl)-1,3,4-oxadiazoles were designed and synthesized as novel benzodiazepine analogues. Most of them revealed similar to superior binding affinity to the GABAA/benzodiazepine receptor complex, relative to diazepam as the reference drug. Among them, 5-(4-chloro-2-(2-fluorophenoxy)benzyl)-3-benzylthio-4H-1,2,4-triazole (8l) showed the highest affinity (IC50=0.892 nM) relative to diazepam (IC50=2.857 nM) and also showed the most increase in pentylenetetrazole-induced seizure threshold relative to diazepam as the reference drug.

Erratum: Review on Approved and Inprogress COVID-19 Vaccines.

[This corrects the article e124228 in vol. 21.].

Optimization of the Synthesis and Radiolabeling of ZIF-8 Nanoparticles.

Background: Lately, there has been increasing interest in the benefits of metal-organic frameworks, and among them, zeolitic imidazolate frameworks (ZIF - 8) stand out as one of the most commonly employed systems owing to their unique characteristics. Objectives: Given that properties like particle size play a key role in biomedical applications of nanoparticles, optimizing the synthesis conditions becomes crucial. Additionally, it is essential to label these nanoparticles to track them effectively within the body. Methods: Zeolitic imidazolate frameworks nanoparticles were synthesized under various conditions, including high and room temperature, using two different solvents: Water and methanol. Modifications were made to the reaction temperature and the ratio of reactants to improve the outcomes. Particle size and size distribution were assessed in all conditions. Additionally, the radiolabeling of nanoparticles was examined using four different methods to identify the method with the highest efficiency and radiochemical purity. Results: The optimum conditions for ZIF-8 synthesis were determined at 50°C using methanol as the solvent. A reactant weight ratio of 1: 2 (zinc nitrate to 2-methylimidazole) was utilized. The most effective radiolabeling approach involved using tin chloride as a reducing agent, with the reaction mixture maintained at a temperature of 70°C for 30 minutes. Conclusions: In this study, the optimum conditions were successfully identified for synthesizing and labeling ZIF-8 nanoparticles. These nanoparticles have the potential to serve as effective carriers for diagnostic and therapeutic agents.

RadioLabeling of the Mutant form of Anti-PlGF Nanobody by 99mTc-Tricarbonyl.

Background: Molecular imaging is a highly effective method for diagnosing cancer and evaluating treatment. A molecular tracer often consists of two segments: A targeting segment, which can be antibodies, antibody fragments, or VHH (nanobody), and a detection segment, such as radioisotopes. The small size of VHH allows for excellent tissue penetration and fast clearance, resulting in minimal nonspecific background, which makes them appealing for use as imaging agents. 99m-technetium (99mTc), one of the well-known radioisotopes, is particularly useful in routine clinical imaging. Objectives: This study aims to construct 99mTc-anti-placenta growth factor (PlGF) nanobody and assess its radiochemical purity (RCP). Methods: The mutant form of anti-PlGF nanobody was expressed in E. coli TG1 and purified using Ni-NTA column affinity chromatography. The purified nanobodies were confirmed by SDS-PAGE and western blotting. A 99mTc-tricarbonyl solution was added to phosphate-buffered saline (PBS) containing the mutant nanobody for labeling, and the mixture was purified using a PD-10 column. Results: The RCP of 99mTc-tricarbonyl is > 98%. After the addition of radioisotopes to the mixture of nanobodies, purity reached 70% in 2 hours and remained constant during incubation. After purifying the labeled nanobody, activity was measured, and the highest amount of labeled nanobodies was collected in the second part. The stability of the labeled nanobody in PBS and in competition with histidine for 4 hours was checked by thin-layer chromatography (TLC). Conclusions: The findings of this study reveal that the RCP of the labeled nanobody was above 95% after 4 hours, indicating the labeled antibody's stability. These results are promising and could be utilized in future in vitro and in vivo studies.

Novel 2-substituted-5-(4-chloro-2-phenoxy)phenyl-1,3,4-oxadiazole derivatives, ligands of GABAA/benzodiazepine receptor complex: Design, synthesis, radioligand binding assay, and pharmacological evaluation.

Agonists of Benzodiazepine (BZD) receptor are exhaustively used in the control of muscle spasms, seizure, anxiety, and insomnia. BZDs have some unwanted effects; therefore, the development of new BZD receptor agonists with better efficacy and fewer unwanted effects is one of the subjects of interest. In this study, based on the pharmacophore/receptor model of the BZD binding site of GABAA receptors, a series of new 2-substituted-5-(4-chloro-2-phenoxy)phenyl-1,3,4-oxadiazole derivatives (6a-f) were designed. Energy minima conformers of the designed compounds and diazepam were well matched in conformational analysis and showed proper interaction with the BZD-binding site of the GABAA receptor model (α1ß2ϒ2) in docking studies. The designed compounds were synthesized in acceptable yield and evaluated for their in vitro affinity to the benzodiazepine receptor of rat brains by radioligand receptor binding assay. The results demonstrated that the affinities of most of the novel compounds were even higher than diazepam. The novel compound 6a with the best affinity in radioligand receptor binding assay (Ki=0.44 nM and IC50= 0.73±0.17 nM) had considerable hypnotic activity and weak anticonvulsant and anxiolytic effects with no negative effect on memory in animal models. Flumazenil as a selective benzodiazepine receptor antagonist was able to prevent hypnotic and anticonvulsant effects of 6a indicating the role of BZD receptors in these effects.

Synthesis and Radiolabeling of Glu-Urea-Lys with 99mTc-Tricarbonyl-Imidazole-Bathophenanthroline Disulfonate Chelation System and Biological Evaluation as Prostate-Specific Membrane Antigen Inhibitor.

Background: The Glu-Urea-Lys (EUK) pharmacophore as prostate-specific membrane antigen (PSMA)-targeted ligand was synthesized, radiolabeled with 99mTc-tricarbonyl-imidazole-BPS chelation system, and biological activities were evaluated. The strategy [2 + 1] ligand is applied for tricarbonyl labeling. (5-imidazole-1-yl)pentanoic acid as a monodentate ligand and bathophenanthroline disulfonate (BPS) as a bidentate ligand formed a chelate system with 99mTc-tricarbonyl. EUK-pentanoic acid-imidazole and EUK were evaluated for PSMA active site using AutoDock 4 software. Materials and Methods: EUK-pentanoic acid-imidazole was synthesized in two steps. BPS was radiolabeled with 99mTc-tricarbonyl at 100°C for 30 min. The purified 99mTc(CO)3(H2O)BPS was used to radiolabel EUK-pentanoic acid-imidazole at 100°C, 30 min. Radiochemical purity, Log P, and stability studies were carried out within 24 h. Affinity of 99mTc(CO)3BPS-imidazole-EUK was performed in the saturation binding studies using LNCaP cells at 37°C for 1 h with a range of 0.001-1000 nM radiolabeled compound range. Internalization studies were performed in LNCaP cells with 1000 nM radiolabeled compound incubated for (0-2) h at 37°C. Biodistribution was studied in normal male Balb/c mice. The artificial intelligence predicts the uptake of radiolabeled compound in tumor. Results: The structures of synthesized compounds were confirmed by mass spectroscopy. Radiochemical purity, Log P, and protein binding were ≥95%, -0.2%, and 23%, respectively. The radiolabeled compound was stable in saline and human plasma within 24 h with radiochemical purity ≥90%. There was no release of 99mTc within 4 h in competition with histidine. The affinity was 82 ± 26.38 nM, and the activity increased inside the cells over time. Biodistribution studies showed radioactivity accumulation in kidneys less than 99mTc-HYNIC-PSMA. There was a moderate accumulation of radioactivity in the liver and intestine. Conclusion: Based on the results, 99mTc(CO)3BPS-imidazole-EUK can potentially be used as an imaging agent for studies at prostate bed and distal areas. The chelate system can be potentially labeled with rhenium for imaging studies (fluorescent or scintigraphy) and therapy.

Synthesis, Structural Characterization, and Cytotoxic Activity of New Benzo[d]imidazo[2,1-b]thiazole Derivatives Against MCF-7 Breast Cancer Cells.

Breast cancer is an invasive disease with a high prevalence among females. Despite various treatments, studies are still ongoing to find an effective treatment for this disease. This study aimed to synthesize a new series of diaryl benzo[d]imidazo[2,1-b]thiazole compounds containing aminoethoxy side chain and in vitro investigate their cytotoxicity on a human breast cancer cell line (MCF-7). Twelve derivatives (6a-6l) were synthesized from this scaffold, the structures of which were spectroscopically confirmed. The cytotoxic effects of the derivatives on the MCF-7 cell line were also assessed using the MTT assay. All these compounds showed a good inhibitory effect on the MCF-7 cell line, compared to that of tamoxifen. Compounds (6i) and (6j) showed higher cytotoxicity with relevant inhibitory effects of 81% and 73%, respectively.

Doxorubicin-Loaded Multivesicular Liposomes (DepoFoam) as a Sustained Release Carrier Intended for Locoregional Delivery in Cancer Treatment: Development, Characterization, and Cytotoxicity Evaluation.

Background: Despite the advantages of direct intratumoral (IT) injection, the relatively rapid withdrawal of most anti-cancer drugs from the tumor due to their small molecular size limits the effectiveness of this method of administration. To address these limitations, recently, increasing attention has been directed to using slow-release biodegradable delivery systems for IT injection. Objectives: This study aimed to develop and characterize a doxorubicin-loaded DepoFoam system as an efficient controlled-release carrier to be employed for locoregional drug delivery in cancer treatment. Methods: Major formulation parameters, including the molar ratio of cholesterol to the main lipid [Chol/egg phosphatidylcholine (EPC)], triolein (TO) content, and lipid-to-drug molar ratio (L/D), were optimized using a two-level factorial design approach. The prepared batches were evaluated for encapsulation efficiency (EE) and percentage of drug release (DR) after 6 and 72 hours as dependent variables. The optimum formulation (named DepoDOX) was further evaluated in terms of particle size, morphology, zeta potential, stability, Fourier-transform infrared spectroscopy, in vitro cytotoxicity, and hemolysis. Results: The analysis of factorial design indicated that TO content and L/D ratio had a negative effect on EE; between these two, TO content had the greatest effect. The TO content was also the most significant component, with a negative effect on the release rate. The ratio of Chol/EPC showed a dual effect on the DR rate. Using a higher percentage of Chol slowed down the initial release phase of the drug; nevertheless, it accelerated the DR rate in the later slow phase. DepoDOX were spherical and honeycomb-like structures (≈ 9.81 µm) with a desired sustained release profile, as DR lasted 11 days. Its biocompatibility was confirmed by the results of cytotoxicity and hemolysis assays. Conclusions: The in vitro characterization of optimized DepoFoam formulation demonstrated its suitability for direct locoregional delivery. DepoDOX, as a biocompatible lipid-based formulation, showed appropriate particle size, high capability for encapsulating doxorubicin, superior physical stability, and a markedly prolonged DR rate. Therefore, this formulation could be considered a promising candidate for locoregional drug delivery in cancer treatment.

Piperine-loaded electrospun nanofibers, an implantable anticancer controlled delivery system for postsurgical breast cancer treatment.

Tumorectomy followed by radiotherapy, hormone, and chemotherapy, are the current mainstays for breast cancer treatment. However, these strategies have systemic toxicities and limited treatment outcomes. Hence, there is a crucial need for a novel controlled release delivery system for implantation following tumor resection to effectively prevent recurrence. Here, we fabricated polycaprolactone (PCL)-based electrospun nanofibers containing piperine (PIP), known for chemopreventive and anticancer activities, and also evaluated the impact of collagen (Coll) incorporation into the matrices. In addition to physicochemical characterization such as morphology, hydrophilicity, drug content, release properties, and mechanical behaviors, fabricated nanofibers were investigated in terms of cytotoxicity and involved mechanisms in MCF-7 and 4T1 breast tumor cell lines. In vivo antitumor study was performed in 4T1 tumor-bearing mice. PIP-PCL75-Coll25 nanofiber was chosen as the optimum formulation due to sustained PIP release, good mechanical performance, and superior cytotoxicity. Demonstrating no organ toxicity, animal studies confirmed the superiority of locally administered PIP-PCL75-Coll25 nanofiber in terms of inhibition of growth tumor, induction of apoptosis, and reduction of cell proliferation compared to PIP suspension, blank nanofiber, and the control. Taken together, we concluded that PIP-loaded nanofibers can be introduced as a promising treatment for implantation upon breast tumorectomy.

Optimization binding studies of opioid receptors, saturation and competition, using [3H]-DAMGO.

BACKGROUND: Opioid analgesics are prescribed for the moderate to severe pain in the clinic. New analogs of µ-opioid receptors are introduced because they may have less adverse effects and better efficacy. However, these new analogs have to be screened for their receptor affinity before entering clinical trial phases. A common method to do such screening is using radioligand-binding-assay, which is a fast and precise screening technique if the assays are done at an optimum condition. One of the main challenges in this type of screening is to separate free/unbound radioligands from bound radioligands. In this study, we applied a centrifugation method instead of a filtration method to separate free radioligands from bound radioligands, and also optimized the conditions for radioligand receptor binding studies of µ-opioid receptors, saturation, and the competition. METHODS: We used the midbrain and brainstem of naltrexone-treated rats as a source of µ-opioid receptors, and [3H]-DAMGO as the radioligand. Naloxone was also used to determine non-specific binding. A given amount of membrane protein was incubated with an increasing amount of radioligand at 37 °C to saturate the receptors at equilibrium and the amount of radioligand saturated in the receptors were used in competition studies. RESULTS: 160 µg membrane protein saturated with 20 nM [3H]-DAMGO at 37 °C for 35 min with Kd (15.06 nM, 95% CI 8.117-22.00) and Bmax (0.4750 pmol/mg, 95% CI 0.3839-0.5660). CONCLUSION: Applying the centrifugation method instead of the filtration to separate free from bound radioligand produced repeatable and reliable results. The optimum conditions for radioligand binding were used in competition studies which resulted in the expected outcomes.