Biodistribution Profile Research Articles

Sensitive Positron Emission Tomography Imaging of PD-L1 Expression in Human Breast and Lung Carcinoma Xenografts Using the Radiometalated Peptide Ga-68-TRAP-WL12.

Noninvasive imaging of the immune checkpoint protein programmed death ligand 1 (PD-L1; synonyms: CD274, B7-H1) holds great promise to improve patient selection and, thus, response rates for immune checkpoint therapy (ICT) with monoclonal antibodies targeting the PD1/PD-L1 axis. The PD-L1 specific peptide WL12 (cyclo(AcY-(NMe)A-N-P-H-L-Hyp-W-S-W(Me)-(NMe)Nle-(NMe)Nle-O-C)-G-NH2) was functionalized with the Gallium-68 chelator TRAP by means of click chemistry (CuAAC). The resulting conjugate TRAP-WL12 was labeled with Gallium-68 within 16 min, with approximately 90% radiochemical yield and 99% radiochemical purity, affording Ga-68-TRAP-WL12 with molar activities typically exceeding 100 MBq/nmol. This radiotracer was characterized by positron emission tomography (PET) imaging and ex vivo biodistribution in murine xenografts of nontransfected PD-L1 expressing tumor cell lines, MDA-MB-231 (human breast carcinoma), and H2009 (human lung adenocarcinoma). It showed a favorable biodistribution profile with rapid renal clearance and low background (tumor-to-blood ratio = 26.6, 3 h p.i.). Conjugation of the Ga-68-TRAP moiety to WL12 successfully mitigated the nonspecific uptake of this peptide in organs, particularly the liver. This was demonstrated by comparing Ga-68-TRAP-WL12 with the archetypical Ga-68-DOTA-WL12, for which tumor-to-liver ratios of 1.4 and 0.5, respectively, were found. Although immunohistochemistry (IHC) revealed a low PD-L1 expression in MDA-MB-213 and H2009 xenografts that corresponds well to the clinical situation, PET showed high tumor uptakes (6.6 and 7.3% injected activity per gram of tissue (iA/g), respectively) for Ga-68-TRAP-WL12. Thus, this tracer has the potential for routine clinical PD-L1 PET imaging because it detects even very low PD-L1 expression densities with high sensitivity and may open an avenue to replace PD-L1 IHC of biopsies as the standard means to select potential responders for ICT.

Assessment of bioactive peptides derived from laminin-111 as prospective breast cancer-targeting agents

Breast cancer remains a pressing public health issue primarily affecting women. Recent research has spotlighted bioactive peptides derived from laminin-111, implicated in breast tumor development. Remarkably, the sequences IKVAV, YIGSR, and KAFDITYVRLKF from the α1, β1, and γ1 chains, respectively, have garnered significant attention. This study aims to assess the potential of these radiolabeled peptides as targeting agents for breast cancer. The three peptides were synthesized using the Fmoc strategy, purified via reversed-phase high-performance liquid chromatography (RP-HPLC), and characterized through mass spectrometry. Iodine-131 (131I) radiolabeling was performed using the chloramine T method, exhibiting high radiochemical yield and stability for [131I]I-YIKVAV and [131I]I-YIGSR. Conversely, [131I]I-KAFDITYVRLKF demonstrated low radiochemical yield and stability and was excluded from the biological studies. The lipophilicity of the compounds ranged from − 2.12 to − 1.10. Serum protein binding assay for [131I]I-YIKVAV and [131I]I-YIGSR reached ≅ 48% and ≅ 25%, respectively. Affinity for breast cancer cells was evaluated using MDA-MB-231 and MCF-7 tumor cell lines, indicating the affinity of the radiopeptides with these tumor cells. Ex vivo biodistribution profiles of the radiopeptides were assessed in the MDA-MB-231 breast tumor animal model, revealing tumor tissue accumulation, supported by a high tumor-to-contralateral muscle ratio and autoradiography. These results signify the effective penetration of YIKVAV and YIGSR into tumor tissue. Therefore, the synthesized α1 and β1 peptide fragments exhibit favorable characteristics as potential breast cancer-targeting agents, promising future exploration as radiopharmaceuticals for breast cancer.

Branched pegylated linker-auristatin to control hydrophobicity for the production of homogeneous minibody-drug conjugate against HER2-positive breast cancer

Trastuzumab emtansine (Kadcyla®) was the first antibody-drug conjugate (ADC) approved by the Food and Drug Administration in 2013 against a solid tumor, and the first ADC to treat human epidermal growth factor receptor 2 positive (HER2+) breast cancer. However, this second generation ADC is burden by several limitations included heterogeneity, limited activity against heterogeneous tumor (regarding antigen expression) and suboptimal tumor penetration. To address this, different development strategies are oriented towards homogeneous conjugation, new drugs, optimized linkers and/or smaller antibody formats. To reach better developed next generation ADCs, a key parameter to consider is the management of the hydrophobicity associated with the linker-drug, increasing with and limiting the drug-to-antibody ratio (DAR) of the ADC. Here, an innovative branched pegylated linker was developed, to control the hydrophobicity of the monomethyl auristatin E (MMAE) and its cathepsin B-sensitive trigger. This branched pegylated linker-MMAE was then used for the efficient generation of internalizing homogeneous ADC of DAR 8 and minibody-drug conjugate of DAR 4, targeting HER2. Both immunoconjugates were then evaluated in vitro and in vivo on breast cancer models. Interestingly, this study highlighted that the minibody-MMAE conjugate of DAR 4 was the best immunoconjugate regarding in vitro cellular internalization and cytotoxicity, gamma imaging, ex vivo biodistribution profile in mice and efficient reduction of tumor size in vivo. These results are very promising and encourage us to explore further fragment-drug conjugate development.

Intradermal delivery of the antiretroviral drugs cabotegravir and rilpivirine by dissolving microarray patches: Investigation of lymphatic uptake

The lymphatic system possesses the main viral replication sites in the body following viral infection. Unfortunately, current antiretroviral agents penetrate the lymph nodes insufficiently when administered orally and, therefore, cannot access the lymphatic system sufficiently to interrupt this viral replication. For this reason, novel drug delivery systems aimed at enhancing the lymphatic uptake of antiretroviral drugs are highly desirable. Dissolving polymeric microarray patches (MAPs) may help to target the lymph intradermally. MAPs are intradermal drug delivery systems used to deliver many types of compounds. The present work describes a novel work investigating the lymphatic uptake of two anti-HIV drugs: cabotegravir (CAB) and rilpivirine (RPV) when delivered intradermally using dissolving MAPs containing nanocrystals of both drugs. Maps were formulated using NCs obtained by solvent-free milling technique. The polymers used to prepare the NCs of both drugs were PVA 10 Kda and PVP 58 Kda. Both NCs were submitted to the lyophilization process and reconstituted with deionized water to form the first layer of drug casting. Backing layers were developed for short application times and effective skin deposition. In vivo biodistribution profiles of RPV and CAB after MAP skin application were investigated and compared with the commercial intramuscular injection using rats. After a single application of RPV MAPs, a higher concentration of RPV was delivered to the axillary lymph nodes (AL) (Cmax 2466 ng/g – Tmax 3 days) when compared with RPV IM injection (18 ng/g - Tmax 1 day), while CAB MAPs delivered slightly lower amounts of drug to the AL (5808 ng/g in 3 days) when compared with CAB IM injection (9225 ng/g in 10 days). However, CAB MAPs delivered 7726 ng/g (Tmax 7 days) to the external lumbar lymph nodes, which was statistically equivalent to IM delivery (Cmax 8282 ng/g - Tmax 7 days). This work provides strong evidence that MAPs were able to enhance the delivery of CAB and RPV to the lymphatic system compared to the IM delivery route.

Controllable synthesis of barium carbonate nano- and microparticles for SPECT and CT imaging.

The design and synthesis of nano- and microcarriers for preclinical and clinical imaging are highly attractive due to their unique features, for example, multimodal properties. However, broad translation of these carriers into clinical practice is postponed due to the unknown biological reactivity of the new components used for their synthesis. Here, we have developed microcarriers (∼2-3 μm) and nanocarriers (<200 nm) made of barium carbonate (BaCO3) for multiple imaging applications in vivo. In general, barium in the developed carriers can be used for X-ray computed tomography, and the introduction of a diagnostic isotope (99mTc) into the BaCO3 structure enables in vivo visualization using single-photon emission computed tomography. The bioimaging has shown that the radiolabeled BaCO3 nano- and microcarriers had different biodistribution profiles and tumor accumulation efficiencies after intratumoral and intravenous injections. In particular, in the case of intratumoral injection, all the types of used carriers mostly remained in the tumors (>97%). For intravenous injection, BaCO3 microcarriers were mainly localized in the lung tissues. However, BaCO3 NPs were mainly accumulated in the liver. These results were supported by ex vivo fluorescence imaging, direct radiometry, and histological analysis. The BaCO3-based micro- and nanocarriers showed negligible in vivo toxicity towards major organs such as the heart, lungs, liver, kidneys, and spleen. This study provides a simple strategy for the design and fabrication of the BaCO3-based carriers for the development of dual bioimaging.

Targeted Nano-Based Systems for the Anti-Obesity Agent's Delivery.

Obesity is a global health concern and a chronic disease that is accompanied by excessive fat storage in adipose and nonadipose tissues. An increase in the body-mass index (BMI) is directly proportional to the 2- to 3.9-fold increase in all-cause mortality in obesity. If left untreated for a longer period, obesity-related metabolic, cardiovascular, inflammatory, and malignant diseases reduce life expectancy. Currently, most of the anti-obesity drugs have failed and fallen into disrepute, either due to their ineffectiveness or adverse effects. In this review, depending on their enhanced pharmacokinetic and biodistribution profiles, whether nanocarriers alter the basic properties and bioactivity of anti-obesity drugs used in clinical practice are debated. First, nanocarriers can improve the safety of still-used anti-obesity drugs by lowering their systemic toxicity through increasing targeting efficacy and preventing drug carrier toxicity. Second, when the micro-ribonucleic acids (miRNAs), which are aberrantly expressed in obesity and obesity-related diseases, are encapsulated into nanoparticles, they are effective in multiple obesity-related metabolic pathways and gene networks. Finally, a synergistic anti-obesity effect with low dose and low toxicity can be obtained with the combinatory therapy applied by encapsulating the anti-obesity drug and gene in the same nanocarrier delivery vehicle.

Assessing inorganic nanoparticle toxicity through omics approaches.

In the last two decades, the development of nanotechnology has resulted in inorganic nanoparticles playing crucial roles in key industries, ranging from healthcare to energy technologies. For instance, gold and silver nanoparticles are widely used in rapid COVID-19 and flu tests, titania and zinc oxide nanoparticles are commonly found in cosmetic products, and superparamagnetic iron oxide nanoparticles have been clinically exploited as contrast agents and anti-anemia medicines. As a result, human exposure to nanomaterials is continuously increasing, raising concerns about their potential adverse health effects. Historically, the study of nanoparticle toxicity has largely relied on macroscopic observations obtained in different in vitro and in vivo models, resulting in readouts such as median lethal dose, biodistribution profile, and/or histopathological assessment. In recent years, omics methodologies, including transcriptomics, epigenomics, proteomics, metabolomics, and lipidomics, are increasingly used to characterize the biological interactions of nanomaterials, providing a better and broader understanding of their impact and mechanisms of toxicity. These approaches have been able to identify important genes and gene products that mediate toxicological effects, as well as endogenous functions and pathways dysregulated by nanoparticles. Omics methods improve our understanding of nanoparticle biology, and unravel mechanistic insights into nanomedicine-based therapies. This review aims to provide a deeper understanding and new perspectives of omics approaches to characterize the toxicity and biological interactions of inorganic nanoparticles, and improve the safety of nanoparticle applications.

PYTA: a universal chelator for advancing the theranostic palette of nuclear medicine.

To clinically advance the growing arsenal of radiometals available to image and treat cancer, chelators with versatile binding properties are needed. Herein, we evaluated the ability of the py2[18]dieneN6 macrocycle PYTA to interchangeably bind and stabilize 225Ac3+, [177Lu]Lu3+, [111In]In3+ and [44Sc]Sc3+, a chemically diverse set of radionuclides that can be used complementarily for targeted alpha therapy, beta therapy, single-photon emission computed tomography (SPECT) imaging, and positron emission tomography (PET) imaging, respectively. Through NMR spectroscopy and X-ray diffraction, we show that PYTA possesses an unusual degree of flexibility for a macrocyclic chelator, undergoing dramatic conformational changes that enable it to optimally satisfy the disparate coordination properties of each metal ion. Subsequent radiolabeling studies revealed that PYTA quantitatively binds all 4 radiometals at room temperature in just minutes at pH 6. Furthermore, these complexes were found to be stable in human serum over 2 half-lives. These results surpass those obtained for 2 state-of-the-art chelators for nuclear medicine, DOTA and macropa. The stability of 225Ac-PYTA and [44Sc]Sc-PYTA, the complexes having the most disparity with respect to metal-ion size, was further probed in mice. The resulting PET images (44Sc) and ex vivo biodistribution profiles (44Sc and 225Ac) of the PYTA complexes differed dramatically from those of unchelated [44Sc]Sc3+ and 225Ac3+. These differences provide evidence that PYTA retains this size-divergent pair of radionuclides in vivo. Collectively, these studies establish PYTA as a new workhorse chelator for nuclear medicine and warrant its further investigation in targeted constructs.

Chelation chemistry of manganese-52 for PET imaging applications

IntroductionDue to its decay and chemical properties, interest in manganese-52 has increased for development of long-lived PET radiopharmaceuticals. Its long half-life of 5.6 days, low average positron energy (242 keV), and sufficient positron decay branching ratio make it suitable for radiolabeling macromolecules for investigating slow biological processes. This work aims to establish suitable chelators for manganese-52 that can be radiolabeled at mild conditions through the evaluation of commercially available chelators. MethodsManganese-52 was produced through the nuclear reaction NatCr(p,n)52Mn by irradiation of natural chromium targets on a TR24 cyclotron followed by purification through ion exchange chromatography. The radiolabeling efficiencies of chelators: DOTA, DiAmsar, TETA, DO3A, NOTA, 4′-Formylbenzo-15-crown-5, Oxo-DO3A, and DFO, were assessed by investigating the impact of pH, buffer type, and temperature. In vitro stability of [52Mn]Mn(DO3A)−, [52Mn]Mn(Oxo-DO3A)−, and [52Mn]Mn(DOTA)2− were evaluated in mouse serum. The radiocomplexes were also evaluated in vivo in mice. Crystals of [Mn(Oxo-DO3A)]− were synthesized by reacting Oxo-DO3A with MnCl2 and characterized by single crystal X-ray diffraction. ResultsYields of 185 ± 19 MBq (5.0 ± 0.5 mCi) (n = 4) of manganese-52 were produced at the end of a 4 h, 15 μA, bombardment with 12.5 MeV protons. NOTA, DO3A, DOTA, and Oxo-DO3A chelators were readily radiolabeled with >96 % radiochemical purity at all conditions. Manganese radiocomplexes of Oxo-DO3A, DOTA, and DO3A remained stable in vitro up to 5 days and exhibited different biodistribution profiles compared to [52Mn]MnCl2. The solid-state structure of Mn-Oxo-DO3A complex was determined by single-crystal X-ray diffraction. ConclusionsDO3A and Oxo-DO3A are suitable chelators for manganese-52 which are readily radiolabeled at mild conditions with high molar activity, and demonstrate both in vitro and in vivo stability.

Specifics of Pharmacokinetics and Biodistribution of 5-Fluorouracil Polymeric Complex.

Monomeric methacrylic acid was used to synthesize polymers; 2-phenylpropane-2-ilbenzodithioate was used for the synthesis of poly(methacrylic acid). To study the molecular-weight characteristics of poly(methacrylic acid) by gel permeation chromatography, an experimental neoplasm model was obtained by grafting PC-1 cancer cells. Blood samples were drawn from the tail vein at different points in time. The rats were sacrificed via decapitation after drawing the last pharmacokinetic blood sample. To study the biodistribution, internal organs were isolated and analyzed. The measurements were carried out by high-performance liquid chromatography. Our results demonstrate that incorporation in a polymeric complex changes the pharmacokinetics and biodistribution profile of 5-FU. The polymeric complex was shown to accumulate to a higher level in the lung and spleen. The results obtained are the basis for further studies to verify the efficacy of the 5-Fluorouracil polymeric complex.

Evaluation of the impact of repeated intravenous phage doses on mammalian host-phage interactions.

Phage therapy has shown great promise for the treatment of multidrug-resistant bacterial infections. However, the lack of a thorough and organized understanding of phage-body interactions has limited its clinical application. Here, we administered different purified phages (Salmonella phage SE_SZW1, Acinetobacter phage AB_SZ6, and Pseudomonas phage PA_LZ7) intravenously to healthy animals (rats and monkeys) to evaluate the phage-induced host responses and phage pharmacokinetics with different intravenous (IV) doses in healthy animals. The plasma and the organs were sampled after different IV doses to determine the phage biodistribution, phage-induced cytokines, and antibodies. The potential side effects of phages on animals were assessed. A non-compartment model revealed that the plasma phage titer gradually decreased over time following a single dose. Repeated doses resulted in a 2-3 Log10 decline of the plasma phage titer at 5 min compared to the first dose, regardless of the type of phage administered in rats. Host innate immune responses were activated including splenic enlargement following repeated doses. Phage-specific neutralization antibodies in animals receiving phages were detected. Similar results were obtained from monkeys. In conclusion, the mammalian bodies were well-tolerant to the administered phages. The animal responses to the phages and the phage biodistribution profiles could have a significant impact on the efficacy of phage therapy.IMPORTANCEPhage therapy has demonstrated potential in addressing multidrug-resistant bacterial infections. However, an insufficient understanding of phage-host interactions has impeded its broader clinical application. In our study, specific phages were administered intravenously (IV) to both rats and monkeys to elucidate phage-host interactions and evaluate phage pharmacokinetics (PK). Results revealed that with successive IV administrations, there was a decrease in plasma phage concentrations. Concurrently, these administrations elicited both innate and adaptive immune responses in the subjects. Notably, the observed immune responses and PK profiles exhibited variation contingent upon the phage type and the mammalian host. Despite these variations, the tested mammals exhibited a favorable tolerance to the IV-administered phages. This underscores the significance of comprehending these interactions for the optimization of phage therapy outcomes.

In vivo safety and biodistribution profile of Klotho-enhanced human urine-derived stem cells for clinical application

BackgroundUrine-derived stem cells (UDSCs) can be easily isolated from urine and possess excellent stem cell characteristics, making them a promising source for cell therapeutics. Due to their kidney origin specificity, UDSCs are considered a superior therapeutic alternative for kidney diseases compared to other stem cells. To enhance the therapeutic potential of UDSCs, we developed a culture method that effectively boosts the expression of Klotho, a kidney-protective therapeutic factor. We also optimized the Good Manufacturing Practice (GMP) system to ensure stable and large-scale production of clinical-grade UDSCs from patient urine. In this study, we evaluated the in vivo safety and distribution of Klotho-enhanced UDSCs after intravenous administration in accordance with Good Laboratory Practice (GLP) regulations.MethodsMortality and general symptoms were continuously monitored throughout the entire examination period. We evaluated the potential toxicity of UDSCs according to the administration dosage and frequency using clinical pathological and histopathological analyses. We quantitatively assessed the in vivo distribution and retention period of UDSCs in major organs after single and repeated administration using human Alu-based qPCR analysis. We also conducted long-term monitoring for 26 weeks to assess the potential tumorigenicity.ResultsKlotho-enhanced UDSCs exhibited excellent homing potential, and recovered Klotho expression in injured renal tissue. Toxicologically harmful effects were not observed in all mice after a single administration of UDSCs. It was also verified that repeated administration of UDSCs did not induce significant toxicological or immunological adverse effects in all mice. Single and repeated administrated UDSCs persisted in the blood and major organs for approximately 3 days and cleared in most organs, except the lungs, within 2 weeks. UDSCs that remained in the lungs were cleared out in approximately 4–5 weeks. There were no significant differences according to the variation of sex and administration frequency. The tumors were found in the intravenous administration group but they were confirmed to be non-human origin. Based on these results, it was clarified that UDSCs have no tumorigenic potential.ConclusionsOur results demonstrate that Klotho-enhanced UDSCs can be manufactured as cell therapeutics through an optimized GMP procedure, and they can be safely administered without causing toxicity and tumorigenicity.

Abstract A146: FPI-2068: A novel anti-EGFR/cMET, alpha-particle emitting, radioimmunoconjugate for cancer therapy

Abstract Introduction: FPI-2068, a targeted alpha therapeutic (TAT), consists of a humanized EGFR and cMET targeting bispecific antibody (FPI-2053) radiolabeled with actinium-225 [225Ac]. FPI-2068 leverages the EGFR/cMET co-expression in cancer (high vs. low in normal tissues) and internalizing nature of the receptors to deliver radionuclides into tumor cells. The primary tumoricidal mechanism of FPI-2068 is double-strand DNA breaks (DSB) induced by alpha radiation, which lead to cell death. Methods: FPI-2068 consists of FPI-2053 conjugated with a DOTA-chelate and radiolabeled with [225Ac]. FPI-2071 is the lutetium-177 analogue. The binding and internalization of FPI-2071 was studied in vitro using cancer cell lines (HT29 colorectal and H292, H441, H1975, HCC827 lung cancers). In vivo studies were performed in corresponding xenograft models. For biodistribution studies FPI-2071 was injected intravenously into mice and tumor/organ radioactivity was quantified ex vivo at selected timepoints. For therapeutic studies, a single dose of FPI-2068 was administered to groups of mice over a range from 92.5 to 740 kBq/kg. Explanted HT29 and H441 tumors from FPI-2068 treated mice were analyzed by immunoblotting to detect activation of DDR and cell death pathways. Results: FPI-2071 demonstrated in vitro binding to all cell lines tested, followed by internalization and retention of the radioisotope into target cells. The biodistribution profile of FPI-2071 showed low normal organ uptake in all xenograft models tested, while tumor uptake varied. The peak tumor uptake in each model was: H292 (~73 %ID/g), H441 (~38 %ID/g), HT29 (~30 %ID/g), HCC827 (~25 %ID/g) and H1975 (20 %ID/g). Delivery of FPI-2068 to target xenografts resulted in anti-tumor efficacy; treatment was well-tolerated by animals. Sustained tumor regression (&amp;gt;28 days) was generally observed at doses of 370 kBq/kg and 740 kBq/kg across models. Lower doses were tumor growth suppressive. FPI-2068 treatment resulted in a dose-dependent increase in pATM and pRad50 expression indicating activation of DDR pathways in HT29 and H441 xenografts. Formation of DSB (pH2AX) and the induction of apoptosis (cleaved caspase 3) was also observed, consistent with the proposed primary mechanism of action. Conclusions: FPI-2068 demonstrated its anti-tumor efficacy in colorectal and lung tumor xenograft mouse models. Single-dose administration of FPI-2068 led to prolonged tumor regression. FPI-2068 caused activation of the DDR pathway as well as apoptosis, suggesting an inability of the cellular machinery to repair the DNA damage induced by the alpha radiation. These data support initiation of clinical trials in patients with solid tumors. Citation Format: Sadaf Aghevlian, Sean Collens, Julie Metcalf, Natalie Grinshtein, John Forbes, Frank Comer, Darshan Dalal, Puja Sapra, Eric Burak. FPI-2068: A novel anti-EGFR/cMET, alpha-particle emitting, radioimmunoconjugate for cancer therapy [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A146.

Characterization and biodistribution of under-employed gene therapy vector AAV7.

The use of adeno-associated viruses (AAVs) as gene delivery vectors has vast potential for the treatment of many severe human diseases. Over one hundred naturally existing AAV capsid variants have been described and classified into phylogenetic clades based on their sequences. AAV8, AAV9, AAVrh.10, and other intensively studied capsids have been propelled into pre-clinical and clinical use, and more recently, marketed products; however, less-studied capsids may also have desirable properties (e.g., potency differences, tissue tropism, reduced immunogenicity, etc.) that have yet to be thoroughly described. These data will help build a broader structure-function knowledge base in the field, present capsid engineering opportunities, and enable the use of novel capsids with unique properties.

Efficient SARS-CoV-2 infection antagonization by rhACE2 ectodomain multimerized onto the Avidin-Nucleic-Acid-NanoASsembly

Nanodecoy systems based on analogues of viral cellular receptors assembled onto fluid lipid-based membranes of nano/extravescicles are potential new tools to complement classic therapeutic or preventive antiviral approaches. The need for lipid-based membranes for transmembrane receptor anchorage may pose technical challenges along industrial translation, calling for alternative geometries for receptor multimerization. Here we developed a semisynthetic self-assembling SARS-CoV-2 nanodecoy by multimerizing the biotin labelled virus cell receptor -ACE2- ectodomain onto a poly-avidin nanoparticle (NP) based on the Avidin-Nucleic-Acid-NanoASsembly-ANANAS. The ability of the assembly to prevent SARS-CoV-2 infection in human lung cells and the affinity of the ACE2:viral receptor-binding domain (RBD) interaction were measured at different ACE2:NP ratios. At ACE2:NP = 30, 90 % SARS-CoV-2 infection inhibition at ACE2 nanomolar concentration was registered on both Wuhan and Omicron variants, with ten-fold higher potency than the monomeric protein. Lower and higher ACE2 densities were less efficient suggesting that functional recognition between multi-ligand NPs and multi-receptor virus surfaces requires optimal geometrical relationships. In vivo studies in mice showed that the biodistribution and safety profiles of the nanodecoy are potentially suitable for preventing viral infection upon nasal instillation. Viral receptor multimerization using ANANAS is a convenient process which, in principle, could be rapidly adapted to counteract also other viral infections.

Preparation and evaluation of radiolabeled acetaminosalol microspheres: A new potential selective radiotracer for ulcerative colitis early diagnosis.

Acetaminosalol labeling reaction with technetium-99m was optimized, and the radiocomplex was obtained in a high radiochemical yield of 98.9 ± 0.6% and high stability (>30 h). The tracer was characterized, and its binding to the PPARγ receptor was assessed in silico. To reduce radiation exposure to non-target organs and increase accumulation in the colon, the tracer was formulated as pH-sensitive microspheres with a mean particle size of 201 ± 2.1μm, a polydispersity index of 0.18, a 25.3 ± 3.6 zeta potential, and 98.6 ± 0.33% entrapment efficiency. The system suitability was assessed in vivo in normal and ulcerative rats, and the biodistribution profile in the colon showed 56.5 ± 1.4% localization within 4h. Blocking study suggested the selectivity of the tracer to the target receptor. Overall, the reported data encouraged the potential use of the labeled microspheres to target ulcerative colitis.

In Vivo Behavior of Systemically Administered Encapsulin Protein Nanocages and Implications for their use in Targeted Drug Delivery

AbstractEncapsulins, self‐assembling protein nanocages derived from prokaryotes, are promising nanoparticle‐based drug delivery systems (NDDS). However, the in vivo behavior and fate of encapsulins are poorly understood. In this study, the interactions between the model encapsulin from Thermotoga maritima (TmEnc) and key biological barriers encountered by NDDS are probed. Here, a purified TmEnc formulation that exhibits colloidal stability, storability, and blood compatibility is intravenously injected into BALB/c mice. TmEnc has an excellent nanosafety profile, with no abnormal weight loss or gross pathology observed, and only temporary alterations in toxicity biomarkers are detected. Notably, TmEnc demonstrates immunogenic properties, inducing the generation of nanocage‐specific IgM and IgG antibodies, but without any prolonged pro‐inflammatory effects. An absence of antibody cross‐reactivity also suggests immune‐orthogonality among encapsulin systems. Moreover, TmEnc forms a serum‐derived protein corona on its surface which changes dynamically and appears to play a role in immune recognition. TmEnc's biodistribution profile further reveals its sequestration from the blood circulation by the liver and then biodegrades within Kupffer cells, thus indicating clearance via the mononuclear phagocyte system. Collectively, these findings provide critical insights into how encapsulins behave in vivo, thereby informing their future design, modification, and application in targeted drug delivery.

Bioinspired Folic Acid-ADH-PLGA Polymeric Nanoparticles Loaded with Mangiferin and Piperine: A Promising Strategy for Targeted Delivery in Multidrug-Resistant Lung Cancer Cells

The current study aims to develop mangiferin and piperine dual drug-loaded folic acid-ADH-PLGA polymeric nanoparticles with bioinspired folic acid modifications which were effective carriers for site-specific delivery in multidrug-resistant lung cancer cells. Mangiferin and piperine, two pharmacological agents, were encapsulated within intelligent nanoparticles. These nanoparticles were fabricated utilizing folic acid, poly(lactic-co-glycolic acid) (PLGA), adipic dihydrazide (ADH) and pluronic F-68 as constituent materials. The successful fabrication of these intelligent polymeric nanoparticles was confirmed through comprehensive characterization employing various analytical techniques, including differential scanning calorimetry (DSC), Fourier transform infrared spectroscopy (FTIR), nuclear magnetic resonance (NMR) and atomic force microscopy (AFM). The efficacy of the intelligent polymeric nanoparticles against cancer cells was assessed through the evaluation of several parameters and their effectiveness was established utilizing the MTT assay. Furthermore, a hemolytic toxicity test was performed to ascertain whether the nanoparticles exerted any toxic effects on red blood cells. Mangiferin and piperine loaded FAP-M and FAP-P nanoparticles revealed a remarkable potential for inducing cell death. The findings from in vitro drug release assessments, hemolytic toxicity evaluations, bio-distribution analyses, MTT assays and apoptosis assessments exhibited substantial cell cycle phase arrest akin to the standard control. The comprehensive investigation and findings of the internalization process revealed the enhanced anticancer efficacy of mangiferin and piperine encapsulated in fibroblast activation protein-modified (FAP-M) and FAP-P nanoparticles as well as their biodistribution profile.

PEGylation of a Peptide-Based Amphiphilic Delivery Agent and Influence on Protein Delivery to Cells.

The cell membrane is a restrictive biological barrier, especially for large, charged molecules, such as proteins. The use of cell-penetrating peptides (CPPs) can facilitate the delivery of proteins, protein complexes, and peptides across the membrane by a variety of mechanisms that are all limited by endosomal sequestration. To improve CPP-mediated delivery, we previously reported the rapid and effective cytosolic delivery of proteins in vitro and in vivo by their coadministration with the peptide S10, which combines a CPP and an endosomal leakage domain. Amphiphilic peptides with hydrophobic properties, such as S10, can interact with lipids to destabilize the cell membrane, thus promoting cargo internalization or escape from endosomal entrapment. However, acute membrane destabilization can result in a dose-limiting cytotoxicity. In this context, the partial or transient deactivation of S10 by modification with methoxy poly(ethylene glycol) (mPEG; i.e., PEGylation) may provide the means to alter membrane destabilization kinetics, thereby attenuating the impact of acute permeabilization on cell viability. This study investigates the influence of PEGylation parameters (molecular weight, architecture, and conjugation chemistry) on the delivery efficiency of a green fluorescent protein tagged with a nuclear localization signal (GFP-NLS) and cytotoxicity on cells in vitro. Results suggest that PEGylation mostly interferes with adsorption and secondary structure formation of S10 at the cell membrane, and this effect is exacerbated by the mPEG molecular weight. This effect can be compensated for by increasing the concentration of conjugates prepared with lower molecular weight mPEG (5 to ∼20 kDa) but not for conjugates prepared with higher molecular weight mPEG (40 kDa). For conjugates prepared with moderate-to-high molecular weight mPEG (10 to 20 kDa), partial compensation of inactivation could be achieved by the inclusion of a reducible disulfide bond, which provides a mechanism to liberate the S10 from the polymer. Grafting multiple copies of S10 to a high-molecular-weight multiarmed PEG (40 kDa) improved GFP-NLS delivery efficiency. However, these constructs were more cytotoxic than the native peptide. Considering that PEGylation could be harnessed for altering the pharmacokinetics and biodistribution profiles of peptide-based delivery agents in vivo, the trends observed herein provide new perspectives on how to manipulate the membrane permeabilization process, which is an important variable for achieving delivery.

New Thiophenyl-pyrazolyl-thiazole Hybrids as DHFR Inhibitors: Design, Synthesis, Antimicrobial Evaluation, Molecular Modeling, and Biodistribution Studies.

The antibiotic resistance problems constitute a considerable threat to human health worldwide; thus, the discovery of new antimicrobial candidates to conquer this issue is an imperative requirement. From this view, new thiophenyl-pyrazolyl-thiazole hybrids 3-10 were synthesized and screened for their antibacterial efficiency versus Gram - and Gram + bacterial strains compared to the reference drug amoxicillin. It was noticed that the new hybrids displayed significant antibacterial efficacy versus Gram - bacteria, especially against Pseudomonas aeruginosa. Also, all the screened candidates demonstrated a noticeable antifungal effect against Candida albicans (MICs = 3.9-125 μg/mL) relative to fluconazole (MIC = 250 μg/mL). Moreover, the new hybrids were investigated for their antituberculosis potency against Mycobacterium tuberculosis (RCMB 010126). Derivatives 4c, 6b, 8b, 9b, and 10b demonstrated prominent antituberculosis efficiency (MICs = 0.12-1.95 μg/mL) compared with the reference drug isoniazid (MIC = 0.12 μg/mL). The latter derivatives were further assessed for their inhibitory potency versus M. tuberculosis DHFR enzyme. The compounds 4c, 6b and 10b presented a remarkable suppression effect with IC50 values of 4.21, 5.70, and 10.59 μM, respectively, compared to that of trimethoprim (IC50 = 6.23 μM). Furthermore, biodistribution profile using radiolabeling way revealed a perceived uptake of 131I-compound 6b into infection induced models. The docking study for the new hybrids 4c, 6b, 8b, 9b and 10b was performed to illustrate the various binding modes with Mtb DHFR enzyme. In silico ADMET studies for the most potent inhibitors 4c, 6b and 10b were also accomplished to predict their pharmacokinetic and physicochemical features.