Drug Delivery Vectors Research Articles

Interactions of a Water-Soluble Glycofullerene with Glucose Transporter 1. Analysis of the Cellular Effects on a Pancreatic Tumor Model.

In recent years, carbon nanomaterials have been intensively investigated for their possible applications in biomedical studies, especially as drug delivery vehicles. Several surface modifications can modulate the unique molecular structure of [60]fullerene derivatives, as well as their physicochemical properties. For this reason, covalent modifications that would enable a greater water solubilization of the fullerene buckyball have been rapidly investigated. The most exciting applications of fullerene nanomaterials are as drug delivery vectors, photosensitizers in photodynamic therapy (PDT), astransfection or MRI contrast agents, antimicrobials and antioxidants. From these perspectives, the glucose derivatives of [60]fullerene seem to be an interesting carbon nanomaterial for biological studies. It is well-known that cancer cells are characterized by an increased glucose uptake and it has also been previously reported that the glucose transporters (GLUTs) are overexpressed in several types of cancers, which make them attractive molecular targets for many drugs. This study explored the use of a highly water-soluble glycofullerene (called Sweet-C60) in pancreatic cancer studies. Here, we describe the PANC-1 cell proliferation, migration, metabolic activity and glycolysis rate after incubations with different concentrations of Sweet-C60. The final results did not show any influence of the Sweet-C60 on various cancer cellular events and glycolysis, suggesting that synthesized glycofullerene is a promising drug delivery vehicle for treating pancreatic cancer.

New approaches in extracellular vesicle engineering for improving the efficacy of anti-cancer therapies.

Cancer is a disease that evolves continuously with unpredictable outcomes. Although conventional chemotherapy can display significant antitumor effects, the lack of specificity and poor bioavailability remain major concerns in cancer therapy. Moreover, with the advent of novel anti-cancer gene therapies, there is an urgent need for drug delivery vectors capable of bypassing cellular barriers and efficiently transferring therapeutic cargo to recipient cells. A number of drug delivery systems have been proposed to overcome these limitations, but their successful clinical translation has been hampered by the onset of unexpected side effects and associated toxicities. The application of extracellular vesicles (EVs), a class of naturally released, cell-derived particles, as drug delivery vectors presents a breakthrough in nanomedicine, taking into account their biocompatibility and natural role in intercellular communication. Combining the advantageous intrinsic properties of EVs with surface functionalization and the encapsulation of drugs allows for a new class of engineered EVs that serve as effective therapeutic carriers. Here, we describe the various successful approaches involving the application of engineered EVs as bio-derived drug delivery vectors in cancer therapy. The latest and most effective strategies of engineering EVs to improve drug loading, stealth properties and tumour targeting capabilities of EVs are debated. Finally, current obstacles and future perspectives of smart engineered EVs are discussed.

Following cytotoxic nanoconjugates from injection to halting the cell cycle machinery and its therapeutic implications in oral cancer

BackgroundThe concept of personalized therapy has been proven to be a promising approach. A popular technique is to utilize gold nanoparticles (AuNPs) as drug delivery vectors for cytotoxic drugs and small molecule inhibitors to target and eradicate oral cancer cells in vitro and in vivo. Both drug and nanocarrier designs play important roles in the treatment efficacy. In our study, we standardized the nanosystem regarding NPs type, size, surface ligands and coverage percentage leaving only the drugs mode of action as the confounding variable. We propose that similarly constructed nanoparticles (NPs) can selectively leverage different conjugated drugs irrelevant to their original mode of action. If proven, AuNPs may have a secondary role beyond bypassing cancer cell membrane and delivering their loaded drugs.MethodsWe conjugated 5- fluorouracil (5Fu), camptothecin (CPT), and a fibroblast growth factor receptor1-inhibitor (FGFR1i) to gold nanospheres (AuNSs). We followed their trajectories in Syrian hamsters with chemically induced buccal carcinomas.ResultsFlow cytometry and cell cycle data shows that 5Fu- and CPT- induced a similar ratio of S-phase cell cycle arrest as nanoconjugates and in their free forms. On the other hand, FGFR1i-AuNSs induced significant sub-G1 cell population compared with its free form. Despite cell cycle dynamics variability, there was no significant difference in tumor cells’ proliferation rate between CPT-, 5Fu- and FGFR1i- AuNSs treated groups. In our in vivo model, FGFR1i-AuNSs induced the highest tumor reduction rates followed by 5Fu- AuNSs. CPT-AuNSs induced significantly lower tumor reduction rates compared with the 5Fu- and FGFR1i- AuNSs despite showing similar proliferative rates in tumor cells.ConclusionsOur data indicates that the cellular biological events do not predict the outcome seen in our in vivo model. Furthermore, our results suggest that AuNSs selectively enhance the therapeutic effect of small molecule inhibitors such as FGFR1i than potent anticancer drugs. Future studies are required to better understand the underlying mechanism.

Multiscale Co-reconstruction of Lung Architectures and Inhalable Materials Spatial Distribution.

The effective pulmonary deposition of inhaled particulate carriers loaded with drugs is a prerequisite for therapeutic effects of drug delivery via inhalation route. Revealing the sophisticated lung scaffold and intrapulmonary distribution of particles at three‐dimensional (3D), in‐situ, and single‐particle level remains a fundamental and critical challenge for dry powder inhalation in pre‐clinical research. Here, taking advantage of the micro optical sectioning tomography system, the high‐precision cross‐scale visualization of entire lung anatomy is obtained. Then, co‐localized lung‐wide datasets of both cyto‐architectures and fluorescent particles are collected at full scale with the resolution down to individual particles. The precise spatial distribution pattern reveals the region‐specific distribution and structure‐associated deposition of the inhalable particles in lungs, which is undetected by previous methods. Overall, this research delivers comprehensive and high‐resolution 3D detection of pulmonary drug delivery vectors and provides a novel strategy to evaluate materials distribution for drug delivery.

Exosomes are Secreted at Distinct Densities in Human Cancer Cells and are Promising Vectors for Hydrophobic Drug Delivery

Exosomes are cell-derived vesicles less than 200 nm in size containing gene encoding and gene silencing RNA and cytosolic proteins with roles in intercellular communication. Interest in the use of exosomes as targeted drug delivery vehicles has grown since it was shown that they can bind to specific cells and deliver intact genetic material to the cytosol of target cells. We present the detailed isolation protocol which allowed us to isolate secreted vesicles, including exosomes, from human prostate cancer (PC3) and melanoma (M21) cell lines. Transmission electron microscopy (TEM) showed that the isolated vesicles displayed a cup-shaped morphology as well as prolate shapes previously unreported in the literature. Nanoparticle Tracking Analysis (NTA) and western blot assays demonstrated that these vesicles were largely within the 30 - 200 nm size range expected for exosomes and were enriched for CD63, an exosome marker protein. NTA data further revealed discrete vesicle subpopulations with narrowly defined size ranges and consistent ratios of 30 - 200 nm vesicles to vesicles of all sizes secreted from both cell lines. These findings suggest a cellular control mechanism governing the fraction of secreted vesicles within the 30 - 200 nm range. Our work provides novel insights on exosome biogenesis and highlights the potential advantages of exosomes as hydrophobic drug carriers.

Direct entry of cell-penetrating peptide can be controlled by maneuvering the membrane curvature

A biomembrane's role is to be a barrier for interior cytosol from an exterior environment to execute the cell's normal biological functions. However, a water-soluble peptide called cell-penetrating peptide (CPP) has been known for its ability to directly penetrate through the biomembranes into cells (cytolysis) without perturbating cell viability and expected to be a promising drug delivery vector. Examples of CPP include peptides with multiple arginine units with strong cationic properties, which is the key to cytolysis. Here we show the conclusive evidence to support the mechanism of CPP’s cytolysis and way to control it. The mechanism we proposed is attributed to biomembrane’s physicochemical nature as lamellar liquid crystal (Lα). Cytolysis occurs as the temporal and local dynamic phase transitions from Lα to an undulated lamellar with pores called Mesh1. We have shown this phase transfer of Lα composed of dioleoyl-phosphatidylcholine (DOPC) with water by adding oligo-arginine (Rx) as CPP at the equilibrium. Using giant unilamellar vesicle composed of DOPC as a single cell model, we could control the level of cytolysis of CPP (FITC-R8) by changing the curvature of the membrane through osmotic pressure modulation. The cytolysis of CPP utilizes biomembrane's inherent topological and functional flexibility corresponding to the stimuli.

Generation of High-Affinity Aptamer-MIP Hybrid Nanoparticles.

Aptamers and molecularly imprinted polymers (MIPs) are two leading technologies used for the development of protein biomimetics. By combining the two technologies, a new hybrid class of materials can be created, which utilizes the interesting characteristics of both recognition materials, while negating several of their drawbacks. This chapter describes the protocol for the synthesis of aptamer-MIP hybrid nanoparticles. These materials exhibit exceptional affinity (into the nM range) and selectivity for their target template. They can be developed for a wide range of targets, while exhibiting excellent robustness, solubility, and flexibility in use. These are a new class of recognition materials with the potential for use as drug delivery vectors, as well as use within sensing and recognition assays.

Self-assembled polymeric nanocarrier-mediated co-delivery of metformin and doxorubicin for melanoma therapy

Malignant melanoma is a life-threatening form of skin cancer with a low response rate to single-agent chemotherapy. Although combined therapies of metformin (MET) and doxorubicin (DOX) are effective in treating a variety of cancers, including breast cancer, their different physicochemical properties and administration routines reduce the effective co-accumulation of both drugs in tumors. Nanoparticles (NPs) have been demonstrated to potentially improve drug delivery efficiency in cancer therapy of, for example, liver and lung cancers. Hence, in this study, we prepared pH-sensitive, biocompatible, tumor-targeting NPs based on the conjugation of biomaterials, including sodium alginate, cholesterol, and folic acid (FCA). As expected, since cholesterol and folic acid are two essentials, but insufficient, substrates for melanoma growth, we observed that the FCA NPs specifically and highly effectively accumulated in xenograft melanoma tumors. Taking advantage of the FCA NP system, we successfully co-delivered a combination of MET and DOX into melanoma tumors to trigger pyroptosis, apoptosis, and necroptosis (PANoptosis) of the melanoma cells, thus blocking melanoma progression. Combined, the establishment of such an FCA NP system provides a promising vector for effective drug delivery into melanoma and increases the possibility and efficiency of drug combinations for cancer treatment.

Design and Synthesis of a Peptide-Based Glioma-Targeted Drug Delivery Vector gHope2.

In the current chapter, we are detailing the synthesis path of a tumor-targeted, CPP-functionalized chemotherapeutic drug, as well as in vitro validation of the targeting and cell penetrating functionalities of the construct. The design of targeted drug delivery vehicle is based on a new glioma-specific homing peptide that has been conjugated to doxorubicin. Further functionalization with an 18-amino acid cell penetrating peptide pVEC was achieved, a CPP that was chosen because of its high cell penetrating efficacy and low toxicity. The three elements were combined into one drug delivery construct gHope2, and its tumor-homing and cell penetrating activity was demonstrated in human glioma cell line U87.

Synergistic enhancement of tendon-to-bone healing via anti-inflammatory and pro-differentiation effects caused by sustained release of Mg2+/curcumin from injectable self-healing hydrogels.

Poor healing response after rotator cuff reconstruction is multifactorial, with the inflammatory microenvironment and deficiency of stem cell differentiation factors at the lesion site being most relevant. However, there is a lack of effective tissue engineering strategies that can simultaneously exert anti-inflammatory and pro-differentiation effects to promote rotator cuff healing.Methods: In this study, we synthesized and characterized a novel active drug delivery vector that successfully overcame the challenge of simultaneous high-efficiency loading and controlled release of Mg2+ and curcumin. The anti-inflammatory and pro-differentiation effects of the composite hydrogel were evaluated in vitro and in vivo. Moreover, healing of the rotator cuff tendon-to-bone interface was studied by histology, immunofluorescence, and biomechanical tests.Results: The composite hydrogel exhibited excellent biocompatibility and injectability, good adhesiveness, and rapid self-healing. The released curcumin showed obvious anti-inflammatory and antioxidation effects, which protected stem cells and tendon matrix. Furthermore, released Mg2+ promoted stem cell aggregation and chondrogenesis. Moreover, biomechanical tests and histological results of a rat rotator cuff tear model at 8 weeks after surgery indicated that the composite hydrogel significantly enhanced tendon-to-bone healing.Conclusions: The composite hydrogel mediated sustained in situ release of curcumin and Mg2+ to effectively promote rotator cuff tendon-to-bone healing via anti-inflammatory and pro-differentiation effects. Therefore, this composite hydrogel offers significant promise for rotator cuff repair.

Preparation and Preliminary Evaluation of Dual-functional Nanoparticles for MRI and siRNA Delivery.

In order to improve the transfection efficiency of gene vectors and monitor the effect of gene therapy, this study prepared a drug delivery vector with dual functions. The thiourea reaction was used to synthesize polyethyleneimine (PEI, MW: 1.8 kDa) with superparamagnetic iron oxide nanoparticles (SPION) (PEI1800-SPION), and the lipid polycationic gene vector PEI1800-SPION loaded cationic liposome (LP-PEI1800-SPION) was further prepared by ethanol injection method. Agarose gel electrophoresis experiment, cytotoxicity experiment, and In-vitro gene silencing experiment were used to evaluate the vector and screen the optimal prescription of LP-PEI1800-SPION/siRNA. When the weight ratio of LP-PEI1800-SPION to siRNA is 20, the transfection efficiency of the nanoparticles was the highest, and the silencing efficiency of the target protein was the largest. The cytotoxicity of LP-PEI1800-SPION was low; when the concentration was in the range of 1-50 μg/mL, the survival rate of the four types of cells was above 80%. Prussian blue staining experiments and In-vitro MRI imaging experiments showed that cells had significant uptake and imaging capabilities for LP-PEI1800-SPION. In conclusion, the visualized polycationic lipid siRNA delivery vehicle (LP-PEI1800-SPION) was successfully prepared in this experiment, which provides a research basis for further theranostics of liver cancer.

Stimuli-Sensitive Self-Assembled Tubules Based on Lysine-Derived Surfactants for Delivery of Antimicrobial Proteins.

Drug delivery vectors based on amphiphiles have important features such as versatile physicochemical properties and stimuli-responsiveness. Amino acid-based surfactants are especially promising amphiphiles due to their enhanced biocompatibility compared to conventional surfactants. They can self-organize into micelles, vesicles and complex hierarchical structures, such as fibers, twisted and coiled ribbons, and tubules. In this work, we investigated the self-assembly and drug loading properties of a family of novel anionic double-tailed lysine-derived surfactants, with variable degree of tail length mismatch, designated as mLys10 and 10Lysn, where m and n are the number of carbon atoms in the tails. These surfactants form tubular aggregates with assorted morphologies in water that undergo gelation due to dense entanglement, as evidenced by light and electron microscopy. Lysozyme (LZM), an enzyme with antimicrobial properties, was selected as model protein for loading. After the characterization of the interfacial properties and phase behavior of the amphiphiles, the LZM-loading ability of the tubules was investigated, under varying experimental conditions, to assess the efficiency of the aggregates as pH- and temperature-sensitive nanocarriers. Further, the toxicological profile of the surfactants per se and surfactant/LZM hydrogels was obtained, using human skin fibroblasts (BJ-5ta cell line). Overall, the results show that the tubule-based hydrogels exhibit very interesting properties for the transport and controlled release of molecules of therapeutic interest.

Triphenylphosphonium-conjugated glycol chitosan microspheres for mitochondria-targeted drug delivery

To develop an efficient vector for mitochondria-targeted drug delivery, we synthesized triphenylphosphonium (TPP)-modified glycol chitosan polymeric microspheres that had a unique chemical structure with both lipophilic phenyl groups and cationic phosphonium. Notably, TPP can easily pass through the phospholipid bilayer of mitochondria, thereby resulting in specific accumulation of a combined drug molecule in the mitochondria due to the membrane potential between TPP and its membrane. Therefore, TPP has been widely used as a mitochondria-targeting moiety. Triphenylphosphonium-glycol chitosan derivatives (GC-TPP and GME-TPP) with two different degrees of substitution (11% and 36%) were prepared by amidation and Michael addition. The chemical structures of GC-TPP and GME-TPP were characterized by 1H nuclear magnetic resonance and Fourier-transform infrared spectroscopy, and their sizes were measured via field emission scanning electron microscopy and dynamic light scattering. Cellular uptake through flow cytometric analysis and confocal microscopy confirmed that both GC-TPP and GME-TPP were well introduced into cells, targeting the mitochondria. In addition, cytotoxicity testing of the most common cell lines, such as HEK293, HeLa, NIH3T3, and HepG2, indicated the absence of polymer toxicity. To evaluate the carrier effectiveness of TPP for drug delivery, doxorubicin (Dox) was used as an anticancer drug. Confocal microscopy images showed that Dox-loaded GME-TPP accumulated inside cells more than Dox-loaded GC-TPP. The anticancer effects of Dox were also determined by MTT assay, apoptosis/necrosis assay, and three-dimensional spheroids. In summary, the results indicate that GC-TPP and GME-TPP microspheres possess great potential as effective drug delivery carriers.

Environmental control of crack propagation in polymer hydrogels

Hydrogels are highly hydrated polymer networks. The synergistic association of a fluid and an elastic phase is the key of numerous applications of hydrogels as food or cosmetic products, drug delivery vectors, wound dressings, scaffolds for tissue regeneration... Since the natural environment for many of these applications is a wet or liquid one, exchange of fluid or solute may occur via the liquid continuum which exists between the environment and the constitutive solvent. In addition to purely osmotic forces, stresses acting on the network are able to drive solvent flow. This is the basis of poroelasticity, initially studied within the framework of consolidated, fluid saturated rocks. The specificity of hydrogels lies on their high stretchability, which makes extended non-linear elasticity the rule rather than the exception when dealing with fracture mechanics. The association of poro- and non-linear elasticity brings the study of rupture of hydrogels at the forefront of research in mechanics. Along this review, we intend to explore the various ways the environment may affect the nucleation, growth and path stability of a crack in a hydrogel. This goal is pursued from a physicist and experimentalist point of view, with special emphasis on dimensionless relevant parameters and order-of-magnitude estimates. A substantial part of the paper is devoted to an introduction to the specific features of soft gel fracture mechanics. We then try and put forward the wide variety of theoretical and experimental issues relevant to environmental crack control with some tentative insight into tissue engineering and living tissue biomechanics.

Efficient LRP1-Mediated Uptake and Low Cytotoxicity of Peptide L57 InVitro Shows Its Promise as CNS Drug Delivery Vector.

Although an abundance of drug candidates exists which are aimed at the remediation of central nervous system (CNS) disorders, the utility of some are severely limited by their inability to cross the blood brain barrier. Potential drug delivery systems such as the Angiopep family of peptides have shown modest potential; however, there is a need for novel drug delivery candidates that incorporate peptidomimetics to enhance the efficiency of transcytosis, specificity, and biocompatibility. Here, we report on the first invitro cellular uptake and cytotoxicity study of a peptidomimetic, cationic peptide, L57. It binds to cluster 4 of the low-density lipoprotein receptor-related protein 1 (LRP1) receptor which is expressed in numerous cell types, such as brain endothelial cells. We used early-passage-number brain microvascular endothelial cells and astrocytes harvested from rat pup brains that highly express LRP1, to study the uptake of L57 versus Angiopep-7 (A7). Uptake of L57 and A7 showed a concentration-dependent increase, with L57 being taken up to a greater degree than A7 at the same concentration. Additionally, peptide uptake in LRP1-deficient PEA 10cells had greatly reduced uptake. Furthermore, L57 demonstrated excellent cell viability versus A7, showing promise as a potential drug delivery vector for CNS therapeutics.

Mesoporous additive-free vaterite CaCO3 crystals of untypical sizes: From submicron to Giant

Mesoporous CaCO3 crystals of sizes in the range 3–15 μm have attracted significant scientific attention as fully decomposable biocompatible drug delivery vectors. They are also used as sacrificial templates to produce polymer-based microcarriers (capsules, beads) and polymer scaffolds for tissue engineering at biologically friendly conditions. However, fabrication of CaCO3 crystals with the sizes beyond the conventional range without additives is still a challenge. There is a desperate need of sub-micrometre and sub-millimetre biocompatible and easily decomposable vectors for intracellular delivery and design of tailor-made polymer scaffolds, respectively. In this study, additive-free one-step fabrication of vaterite mesoporous crystals with sizes in the range from submicron (0.72 μm) up to tens of microns (55 μm) sizes is proposed. Crystals are grown by mixing of Ca2+ and CO32− salts in aqueous media. Internal structure and morphology of the crystals have been investigated. Encapsulation of lysozyme by means of co-synthesis shows an enhanced entrapment into submicron crystals. Besides, the approach to grow CaCO3 crystals on the surface of microcellulose ьш is demonstrated opening broad avenues for the development of vaterite coatings technologies.

Viburnum nervosum Leaf Extract Mediated Green Synthesis of Silver Nanoparticles: A Viable Approach to Increase the Efficacy of an Anticancer Drug.

There is an urgent need to devise improved alternatives for the efficient delivery of drugs to develop improved therapeutic interventions against cancers. Nanotechnology-based drug delivery vehicles are in-use with obvious issues of toxicity and bio-distribution. Therefore, green synthetic routes are being deployed to replace the conventional nanoparticle formulations for effective drug delivery aiming at developing interventional strategies against cancer. A simple, viable, and fast approach was used for the green synthesis of silver nanoparticles (AgNPs) using aqueous leaf-extract of Viburnum nervosum (VN) and to explore the anti-cancer potential of the crude extract of VN. Silver NPs were synthesized by reacting silver nitrate (AgNO3) with leaf extract of VN. Various analytical techniques were used to characterize the AgNPs. Finally, the anti-cancer potential of VN was observed when delivered through AgNPs. The surface plasmon spectra for AgNPs exhibited absorbance peak at 445 nm, and Fourier-Transform Infrared Spectroscopy investigation revealed the presence of biomolecules acting as an effective reducing and capping agent for converting silver nitrate to AgNPs. Further, our results suggest the spherical size of synthesized AgNPs ranging from 12-17 nm. Moreover, in vitro studies conducted for VN extract with breast (MCF-7) and epidermal carcinoma (A431) cells showed biocompatibility. Doxorubicin loaded AgNPs documented an increased bioavailability of the drug compared to the free drug, suggesting the use of AgNPs as "novel drug delivery vectors".

59 Poster Discussion - NLS designed algorithm to develop peptides with a net-neutral charge as potential next-generation vectors for drug delivery to the nucleus

Background: Active import of proteins in the nucleus is primarily determined by the recognition and binding of nuclear localization signals (NLSs) contained in cargo proteins with specific NLS receptors known as importins. Typically, cargo proteins are bound by the adaptor protein importin-a. Importin-b then binds importin-a and the complex is transported and docks at the nuclear pore complex where the cargo is released into the nucleus. As a result, there is great effort to develop NLS-tagged vectors for numerous therapeutic applications that involve maximizing effectiveness via localization inside the nucleus. Despite these efforts, efficient nuclear localization has been difficult.

Two dimensional porous frameworks of graphyne family as therapeutic delivery vehicles for Idarubicin biomolecule in silico: Density functional theory and molecular dynamics simulation

In the present article, transfer efficiency of graphyne's family members as Idarubicin drug delivery systems is evaluated by a combined density functional theory and molecular dynamics simulation approaches in the liquid water. The density functional theory computations such as Non-Covalent Interaction methodology allow the classification of the interatomic interaction of drug molecule with nanosheet as a weak interaction with the adsorption strength value of −81.305 kJ/mol. The density functional theory calculations approve the physical nature of Idarubicin anti-cancer drug interaction with graphydine nanosheet. It is found that graphydine as a good drug delivery vector can carry and deliver Idarubicin drug into the cells without significant changes in the structural and electronic properties of drug molecule in the delivery process. Furthermore, the dynamic behavior of Idarubicin loading on the graphyne-3 nanosheet is investigated utilizing molecular dynamics simulation. It is observed that drug molecules move toward the nanocarrier at the initial time of simulation and adsorb with the parallel orientation relative to the nanosheet surface. Furthermore, molecular dynamics simulation is applied to explore the effects of electric field (magnitude and direction) on the adsorption of Idarubicin drugs on the nanosheet surface. It is found that when the electric field is introduced into the simulation system in z- and xy-directions, the van der Waals interaction energy value shows the increasing trend. In other words, the interaction between the adsorbed drug and nanosheet can be strengthened by applying the electric fields in z- and xy-directions.

Acute and Chronic Dosing of a High-Affinity Rat/Mouse Chimeric Transferrin Receptor Antibody in Mice

Non-invasive brain delivery of neurotherapeutics is challenging due to the blood-brain barrier. The revived interest in transferrin receptor antibodies (TfRMAbs) as brain drug-delivery vectors has revealed the effect of dosing regimen, valency, and affinity on brain uptake, TfR expression, and Fc-effector function side effects. These studies have primarily used monovalent TfRMAbs with a human constant region following acute intravenous dosing in mice. The effects of a high-affinity bivalent TfRMAb with a murine constant region, without a fusion partner, following extravascular dosing in mice are, however, not well characterized. Here we elucidate the plasma pharmacokinetics and safety of a high-affinity bivalent TfRMAb with a murine constant region following acute and chronic subcutaneous dosing in adult C57BL/6J male mice. Mice received a single (acute dosing) 3 mg/kg dose, or were treated for four weeks (chronic dosing). TfRMAb and control IgG1 significantly altered reticulocyte counts following acute and chronic dosing, while other hematologic parameters showed minimal change. Chronic TfRMAb dosing did not alter plasma- and brain-iron measurements, nor brain TfR levels, however, it significantly increased splenic-TfR and -iron. Plasma concentrations of TfRMAb were significantly lower in mice chronically treated with IgG1 or TfRMAb. Overall, no injection related reactions were observed in mice.