In Vivo Biodistribution, Clearance, and Biocompatibility of Multiple Carbon Dots Containing Nanoparticles for Biomedical Application

Current research on the use of carbon dots for various biological systems mainly focuses on the single carbon dots, while particles that contain multiple carbon dots have scarcely been investigated. Here, we assessed multiple carbon dots-crosslinked polyethyleneimine nanoparticles (CDs@PEI) for their in vivo biodistribution, clearance, biocompatibility, and cellular uptake. The in vivo studies demonstrate three unique features of the CDs@PEI nanoparticles: (1) the nanoparticles possess tumor-targeting ability with steady and prolonged retention time in the tumor region. (2) The nanoparticles show hepatobiliary excretion and are clear from the intestine in feces. (3) The nanoparticles have much better biocompatibility than the polyethyleneimine passivated single carbon dots (PEI-CD). We also found that pegylated CDs@PEI nanoparticles can be effectively taken up by the cells, which the confocal laser scanning microscope can image under different excitation wavelengths (at 405, 488, and 800 nm). These prior studies provide invaluable information and new opportunities for this new type of intrinsic photoluminescence nanoparticles in carbon dot-based biomedical applications.

Discovery, optimization and biodistribution of an Affibody molecule for imaging of CD69

Due to the wide scale of inflammatory processes in different types of disease, more sensitive and specific biomarkers are required to improve prevention and treatment. Cluster of differentiation 69 (CD69) is one of the earliest cell surface proteins expressed by activated leukocytes. Here we characterize and optimize potential new imaging probes, Affibody molecules targeting CD69 for imaging of activated immune cells. Analysis of candidates isolated in a previously performed selection from a Z variant E. coli library to the recombinant extracellular domain of human CD69, identified one cross-reactive Z variant with affinity to murine and human CD69. Affinity maturation was performed by randomization of the primary Z variant, followed by selections from the library. The resulting Z variants were evaluated for affinity towards human and murine CD69 and thermal stability. The in vivo biodistribution was assessed by SPECT/CT in rats following conjugation of the Z variants by a DOTA chelator and radiolabeling with Indium-111. A primary Z variant with a Kd of approximately 50 nM affinity to human and murine CD69 was identified. Affinity maturation generated 5 additional Z variants with improved or similar affinity. All clones exhibited suitable stability. Radiolabeling and in vivo biodistribution in rat demonstrated rapid renal clearance for all variants, while the background uptake and washout varied. The variant ZCD69:4 had the highest affinity for human and murine CD69 (34 nM) as well as the lowest in vivo background binding. In summary, we describe the discovery, optimization and evaluation of novel Affibody molecules with affinity for CD69. Affibody molecule ZCD69:4 is suitable for further development for imaging of activated immune cells.

Electrostatic Coating of Viral Particles for Gene Delivery Applications in Muscular Dystrophies: Influence of Size on Stability and Antibody Protection

Background: Duchenne Muscular Dystrophy (DMD) is one of the most common muscular dystrophies, caused by mutated forms of the dystrophin gene. Currently, the only treatment available is symptoms management. Novel approximations are trying to treat these patients with gene therapy, namely, using viral vectors. However, these vectors can be recognized by the immune system decreasing their therapeutic activity and making impossible a multidose treatment due to the induction of the humoral immunity following the first dose. Objective: Our objective is to demonstrate the feasibility of using a hybrid vector to avoid immune clearance, based on the electrostatic coating of adeno-associated virus (AAVs) vectors with our proprietary polymers. Methods: We coated model adeno-associated virus vectors by electrostatic interaction of our cationic poly (beta aminoester) polymers with the viral anionic capsid and characterized biophysical properties. Once the nanoformulations were designed, we studied their in vivo biodistribution by bioluminescence analysis and we finally studied the capacity of the polymers as potential coatings to avoid antibody neutralization. Results: We tested two polymer combinations and we demonstrated the need for poly(ethylene glycol) addition to avoid vector aggregation after coating. In vivo biodistribution studies demonstrated that viral particles are located in the liver (short times) and also in muscles (long times), the target organ. However, we did not achieve complete antibody neutralization shielding using this electrostatic coating. Conclusions: The null hypothesis stands: although it is feasible to coat viral particles by electrostatic interaction with a proprietary polymer, this strategy is not appropriate for AAVs due to their small size, so other alternatives are required as a novel treatment for DMD patients.

In Vitro and In Vivo Evaluation of Human Adenovirus Type 49 as a Vector for Therapeutic Applications

The human adenovirus phylogenetic tree is split across seven species (A–G). Species D adenoviruses offer potential advantages for gene therapy applications, with low rates of pre-existing immunity detected across screened populations. However, many aspects of the basic virology of species D—such as their cellular tropism, receptor usage, and in vivo biodistribution profile—remain unknown. Here, we have characterized human adenovirus type 49 (HAdV-D49)—a relatively understudied species D member. We report that HAdV-D49 does not appear to use a single pathway to gain cell entry, but appears able to interact with various surface molecules for entry. As such, HAdV-D49 can transduce a broad range of cell types in vitro, with variable engagement of blood coagulation FX. Interestingly, when comparing in vivo biodistribution to adenovirus type 5, HAdV-D49 vectors show reduced liver targeting, whilst maintaining transduction of lung and spleen. Overall, this presents HAdV-D49 as a robust viral vector platform for ex vivo manipulation of human cells, and for in vivo applications where the therapeutic goal is to target the lung or gain access to immune cells in the spleen, whilst avoiding liver interactions, such as intravascular vaccine applications.

In Vitro and In Vivo Evaluation of Human Adenovirus Type 49 as a Vector for Therapeutic Applications

The human adenovirus phylogenetic tree is split across seven species (A-G). Species D adenoviruses offer potential advantages for gene therapy applications, with low rates of preexisting immunity detected across screened populations. However, many aspects of the basic virology of species D, such as their cellular tropism, receptor usage and in vivo biodistribution profile, remain unknown. Here, we have characterized human adenovirus type 49 (HAdV-D49), a relatively understudied species D member. We report that HAdV-D49 does not appear to use a single pathway to gain cell entry but appears able to interact with various surface molecules for entry. As such, HAdV-D49 can transduce a broad range of cell types in vitro, with variable engagement of blood coagulation FX. Interestingly, when comparing in vivo biodistribution to adenovirus type 5, HAdV-D49 vectors show reduced liver targeting whilst maintaining transduction of lung and spleen. Overall, this presents HAdV-D49 as a robust viral vector platform for ex vivo manipulation of human cells and for in vivo applications where the therapeutic goal is to target the lung or gain access to immune cells in the spleen whilst avoiding liver interactions, such as intravascular vaccine applications.

In Vitro and In Vivo Evaluation of Human Adenovirus Type 49 as a Vector for Therapeutic Applications

The human adenovirus phylogenetic tree is split across seven species (A-G). Species D adenoviruses offer potential advantages for gene therapy applications, with low rates of preexisting immunity detected across screened populations. However, many aspects of the basic virology of species D, such as their cellular tropism, receptor usage and in vivo biodistribution profile, remain unknown. Here, we have characterized human adenovirus type 49 (HAdV-D49), a relatively understudied species D member. We report that HAdV-D49 does not appear to use a single pathway to gain cell entry but appears able to interact with various surface molecules for entry. As such, HAdV-D49 can transduce a broad range of cell types in vitro, with variable engagement of blood coagulation FX. Interestingly, when comparing in vivo biodistribution to adenovirus type 5, HAdV-D49 vectors show reduced liver targeting whilst maintaining transduction of lung and spleen. Overall, this presents HAdV-D49 as a robust viral vector platform for ex vivo manipulation of human cells and for in vivo applications where the therapeutic goal is to target the lung or gain access to immune cells in the spleen whilst avoiding liver interactions, such as intravascular vaccine applications.