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Targeted delivery of diagnostics and therapeutics offers essential advantages over nontargeted systemic delivery. These include the reduction of toxicity, the ability to reach sites beyond biological barriers, and the delivery of higher cargo concentrations to diseased sites. Virus-like particles (VLPs) can efficiently be used for targeted delivery purposes. VLPs are derived from the coat proteins of viral capsids. They are self-assembled, biodegradable, and homogeneously distributed. In this study, hepatitis E virus (HEV) VLP derivatives, hepatitis E virus nanoparticles (HEVNPs), were radiolabeled with gallium-68, and consequently, the biodistribution of the labeled [Ga-68]Ga-DOTA-HEVNPs was studied in mice. The results indicated that [Ga-68]Ga-DOTA-HEVNPs can be considered as promising theranostic nanocarriers, especially for hepatocyte-targeting therapies.

Resiquimod (R848) is a toll-like receptor 7 and 8 (TLR7/8) agonist with potent antitumor and immunostimulatory activity. However, systemic delivery of R848 is poorly tolerated because of its poor solubility in water and systemic immune activation. In order to address these limitations, we developed an intravenously-injectable formulation with R848 using thermosensitive liposomes (TSLs) as a delivery vehicle. R848 was remotely loaded into TSLs composed of DPPC: DSPC: DSPE-PEG2K (85:10:5, mol%) with 100mM FeSO4 as the trapping agent inside. The final R848 to lipid ratio of the optimized R848-loaded TSLs (R848-TSLs) was 0.09 (w/w), 10-fold higher than the previously-reported values. R848-TSLs released 80% of R848 within 5min at 42°C. These TSLs were then combined with αPD-1, an immune checkpoint inhibitor, and ultrasound-mediated hyperthermia in a neu deletion (NDL) mouse mammary carcinoma model (Her2+, ER/PR negative). Combined with αPD-1, local injection of R848-TSLs showed superior efficacy with complete NDL tumor regression in both treated and abscopal sites achieved in 8 of 11 tumor bearing mice over 100days. Immunohistochemistry confirmed enhanced CD8+ T cell infiltration and accumulation by R848-TSLs. Systemic delivery of R848-TSLs, combined with local hyperthermia and αPD-1, inhibited tumor growth and extended median survival from 28days (non-treatment control) to 94days. Upon re-challenge with reinjection of tumor cells, none of the previously cured mice developed tumors, as compared with 100% of age-matched control mice. The dose of R848 (10μg for intra-tumoral injection or 6mg/kg for intravenous injection delivered up to 4 times) was well-tolerated without weight loss or organ hypertrophy. In summary, we developed R848-TSLs that can be administered locally or systematically, resulting in tumor regression and enhanced survival when combined with αPD-1 in mouse models of breast cancer.

Targeted delivery of diagnostics and therapeutics offers essential advantages over nontargeted systemic delivery. These include the reduction of toxicity, the ability to reach sites beyond biological barriers, and the delivery of higher cargo concentrations to diseased sites. Virus-like particles (VLPs) can efficiently be used for targeted delivery purposes. VLPs are derived from the coat proteins of viral capsids. They are self-assembled, biodegradable, and homogeneously distributed. In this study, hepatitis E virus (HEV) VLP derivatives, hepatitis E virus nanoparticles (HEVNPs), were radiolabeled with gallium-68, and consequently, the biodistribution of the labeled [

The use of nanoparticulate systems to diagnose and treat tumors has gained momentum with the rapid development of nanomedicine. Many nanotheranostics fail due to insufficient bioavailability and low accumulation at the tumor site, resulting in undesirable side effects. We describe the use of an engineered hepatitis E viral nanoparticle (HEVNP) with enhanced bioavailability, tissue retention and mucosal penetration capacities. HEVNP is a modular nanocapsule that can encapsulate heterologous nucleotides, proteins and inorganic metals, such as ferrite oxide nanoparticles. Additionally, the exterior protruding arms of HEVNP is composed of loops that are used for chemical coupling of targeting and therapeutic peptides. We propose the use of HEVNP to target colorectal cancer (i.e., polyps) with imaging-guided delivery using colonoscopy.

Adeno-associated viruses (AAVs) are typically single-stranded deoxyribonucleic acid (ssDNA) encapsulated within 25-nm protein capsids. Recently, tissue-specific AAV capsids (e.g. PHP.eB) have been shown to enhance brain delivery in rodents via the LY6A receptor on brain endothelial cells. Here, we create a non-invasive positron emission tomography (PET) methodology to track viruses. To provide the sensitivity required to track AAVs injected at picomolar levels, a unique multichelator construct labeled with a positron emitter (Cu-64, t1/2 = 12.7 h) is coupled to the viral capsid. We find that brain accumulation of the PHP.eB capsid 1) exceeds that reported in any previous PET study of brain uptake of targeted therapies and 2) is correlated with optical reporter gene transduction of the brain. The PHP.eB capsid brain endothelial receptor affinity is nearly 20-fold greater than that of AAV9. The results suggest that novel PET imaging techniques can be applied to inform and optimize capsid design., Adeno-associated viruses (AAVs) can be targeted in a tissue-specific manner, but their tissue accumulation cannot be assessed in a non-invasive manner. Here the authors conjugate a multivalent chelator labelled with Cu-64 to the surface of AAVs and image the brain accumulation of the PHB.eB capsid by PET.

Significance Although efforts have been made to determine the structure of talin and the way it interacts with integrins through the “head” domain, our work shows now that many of the previous mechanistic models based on the talin adapter are likely to be misleading as they are constructed on a crystal structure representing an improperly folded talin head domain. In this work, we identified the problem with the current talin head model and proposed a FERM-folded talin head. By analyzing these structural features of the FERM-folded talin head in a cellular context, involving also the kindlin adapter, we are making a critical and unprecedented contribution to the understanding and regulation of cell-matrix adhesions., Binding of the intracellular adapter proteins talin and its cofactor, kindlin, to the integrin receptors induces integrin activation and clustering. These processes are essential for cell adhesion, migration, and organ development. Although the talin head, the integrin-binding segment in talin, possesses a typical FERM-domain sequence, a truncated form has been crystallized in an unexpected, elongated form. This form, however, lacks a C-terminal fragment and possesses reduced β3-integrin binding. Here, we present a crystal structure of a full-length talin head in complex with the β3-integrin tail. The structure reveals a compact FERM-like conformation and a tightly associated N-P-L-Y motif of β3-integrin. A critical C-terminal poly-lysine motif mediates FERM interdomain contacts and assures the tight association with the β3-integrin cytoplasmic segment. Removal of the poly-lysine motif or disrupting the FERM-folded configuration of the talin head significantly impairs integrin activation and clustering. Therefore, structural characterization of the FERM-folded active talin head provides fundamental understanding of the regulatory mechanism of integrin function.

Ex vivo programming of T cells can be efficacious but is complex and expensive; therefore, the development of methods to transfect T cells in situ is important. We developed and optimized anti-CD3-targeted lipid nanoparticles (aCD3-LNPs) to deliver tightly packed, reporter gene mRNA specifically to T cells. In vitro, targeted LNPs efficiently delivered mCherry mRNA to Jurkat T cells, and T-cell activation and depletion were associated with aCD3 antibody coating on the surface of LNPs. aCD3-LNPs, but not non-targeted LNPs, accumulated within the spleen following systemic injection, with mCherry and Fluc signals visible within 30 minutes after injection. At 24 h after aCD3-LNP injection, 2-4% of all splenic T cells and 2-7% of all circulating T cells expressed mCherry, and this was dependent on aCD3 coating density. Targeting and transfection were accompanied by systemic CD25(+), OX40(+), and CD69(+) T-cell activation with temporary CD3e ligand loss and depletion of splenic and circulating subsets. Migration of splenic CD8a(+) T cells from the white-pulp to red-pulp, and differentiation from naïve to memory and effector phenotypes, followed upon aCD3-LNP delivery. Additionally, aCD3-LNP injection stimulated the secretion of myeloid-derived chemokines and T-helper cytokines into plasma. Lastly, we administered aCD3-LNPs to tumor bearing mice and found that transfected T cells localized within tumors and tumor-draining lymph nodes following immunotherapy treatment. In summary, we show that CD3-targeted transfection is feasible, yet associated with complex immunological consequences that must be further studied for potential therapeutic applications.

Journal of cell science 133(19), jcs239202 (1-15) - (2020). doi:10.1242/jcs.239202, Integrin activation and clustering by talin are early steps of cell adhesion. Membrane-bound talin head domain and kindlin bind to the �� integrin cytoplasmic tail, cooperating to activate the heterodimeric integrin, and the talin head domain induces integrin clustering in the presence of Mn$^{2+}$. Here we show that kindlin-1 can replace Mn2+ to mediate ��3 integrin clustering induced by the talin head, but not that induced by the F2���F3 fragment of talin. Integrin clustering mediated by kindlin-1 and the talin head was lost upon deletion of the flexible loop within the talin head F1 subdomain. Further mutagenesis identified hydrophobic and acidic motifs in the F1 loop responsible for ��3 integrin clustering. Modeling, computational and cysteine crosslinking studies showed direct and catalytic interactions of the acidic F1 loop motif with the juxtamembrane domains of ��- and ��3-integrins, in order to activate the ��3 integrin heterodimer, further detailing the mechanism by which the talin���kindlin complex activates and clusters integrins. Moreover, the F1 loop interaction with the ��3 integrin tail required the newly identified compact FERM fold of the talin head, which positions the F1 loop next to the inner membrane clasp of the talin-bound integrin heterodimer., Published by Company of Biologists Limited, Cambridge

Gemcitabine delivery to pancreatic ductal adenocarcinoma is limited by poor pharmacokinetics, dense fibrosis and hypo-vascularization. Activatable liposomes, with drug release resulting from local heating, enhance serum stability and circulation, and the released drug retains the ability to diffuse within the tumor. A limitation of liposomal gemcitabine has been the low loading efficiency. To address this limitation, we used the superior solubilizing potential of copper(II) gluconate to form a complex with gemcitabine at copper:gemcitabine (1:4). Thermosensitive liposomes composed of DPPC:DSPC:DSPE-PEG2k (80:15:5, mole%) then reached 12 weight % loading, 4-fold greater than previously reported values. Cryo transmission electron microscopy confirmed the presence of a liquid crystalline gemcitabine-copper mixture. The optimized gemcitabine liposomes released 60% and 80% of the gemcitabine within 1 and 5 min, respectively, at 42°C. Liposomal encapsulation resulted in a circulation half-life of ~2 h in vivo (compared to reported circulation of 16 min for free gemcitabine in mice), and free drug was not detected within the plasma. The resulting gemcitabine liposomes were efficacious against both murine breast cancer and pancreatic cancer in vitro. Three repeated treatments of activatable gemcitabine liposomes plus ultrasound hyperthermia regressed or eliminated tumors in the neu deletion model of murine breast cancer with limited toxicity, enhancing survival when compared to treatment with gemcitabine alone. With 5% of the free gemcitabine dose (5 rather than 100 mg/kg), tumor growth was suppressed to the same degree as gemcitabine. Additionally, in a more aggressive tumor model of murine pancreatic cancer, liposomal gemcitabine combined with local hyperthermia induced cell death and regions of apoptosis and necrosis.

Catheter-based intra-arterial drug therapies have proven effective for a range of oncologic, neurologic, and cardiovascular applications. However, these procedures are limited by their invasiveness and relatively broad drug spatial distribution. The ideal technique for local pharmacotherapy would be noninvasive and would flexibly deliver a given drug to any region of the body with high spatial and temporal precision. Combining polymeric perfluorocarbon nanoemulsions with existent clinical focused ultrasound systems could in principle meet these needs, but it has not been clear whether these nanoparticles could provide the necessary drug loading, stability, and generalizability across a range of drugs, beyond a few niche applications. Here, we develop polymeric perfluorocarbon nanoemulsions into a generalized platform for ultrasound-targeted delivery of hydrophobic drugs with high potential for clinical translation. We demonstrate that a wide variety of drugs may be effectively uncaged with ultrasound using these nanoparticles, with drug loading increasing with hydrophobicity. We also set the stage for clinical translation by delineating production protocols that are scalable and yield sterile, stable, and optimized ultrasound-activated drug-loaded nanoemulsions. Finally, we exhibit a new potential application of these nanoemulsions for local control of vascular tone. This work establishes the power of polymeric perfluorocarbon nanoemulsions as a clinically-translatable platform for efficacious, noninvasive, and localized ultrasonic drug uncaging for myriad targets in the brain and body.

Virus-like particles (VLPs) have been used as nanocarriers to display foreign epitopes and/or deliver small molecules in the detection and treatment of various diseases. This application relies on genetic modification, self-assembly, and cysteine conjugation to fulfill the tumor-targeting application of recombinant VLPs. Compared with genetic modification alone, chemical conjugation of foreign peptides to VLPs offers a significant advantage because it allows a variety of entities, such as synthetic peptides or oligosaccharides, to be conjugated to the surface of VLPs in a modulated and flexible manner without alteration of the VLP assembly. Here, we demonstrate how to use the hepatitis E virus nanoparticle (HEVNP), a modularized theranostic capsule, as a multifunctional delivery carrier. Functions of HEVNPs include tissue-targeting, imaging, and therapeutic delivery. Based on the well-established structural research of HEVNP, the structurally independent and surface-exposed residues were selected for cysteine replacement as conjugation sites for maleimide-linked chemical groups via thiol-selective linkages. One particular cysteine-modified HEVNP (a Cys replacement of the asparagine at 573 aa (HEVNP-573C)) was conjugated to a breast cancer cell-specific ligand, LXY30 and labeled with near-infrared (NIR) fluorescence dye (Cy5.5), rendering the tumor-targeted HEVNPs as effective diagnostic capsules (LXY30-HEVNP-Cy5.5). Similar engineering strategies can be employed with other macromolecular complexes with well-known atomic structures to explore potential applications in theranostic delivery.

Squalenoylation of gemcitabine, a front-line therapy for pancreatic cancer, allows for improved cellular-level and system-wide drug delivery. The established methods to conjugate squalene to gemcitabine and to form nanoparticles (NPs) with the squalenoylated gemcitabine (SqGem) conjugate are cumbersome, time-consuming and can be difficult to reliably replicate. Further, the creation of multi-functional SqGem-based NP theranostics would facilitate characterization of in vivo pharmacokinetics and efficacy. Methods: Squalenoylation conjugation chemistry was enhanced to improve reliability and scalability using tert-butyldimethylsilyl (TBDMS) protecting groups. We then optimized a scalable microfluidic mixing platform to produce SqGem-based NPs and evaluated the stability and morphology of select NP formulations using dynamic light scattering (DLS) and transmission electron microscopy (TEM). Cytotoxicity was evaluated in both PANC-1 and KPC (KrasLSL-G12D/+; Trp53LSL-R172H/+; Pdx-Cre) pancreatic cancer cell lines. A 64Cu chelator (2-S-(4-aminobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid, NOTA) was squalenoylated and used with positron emission tomography (PET) imaging to monitor the in vivo fate of SqGem-based NPs. Results: Squalenoylation yields of gemcitabine increased from 15% to 63%. Cholesterol-PEG-2k inclusion was required to form SqGem-based NPs using our technique, and additional cholesterol inclusion increased particle stability at room temperature; after 1 week the PDI of SqGem NPs with cholesterol was ~ 0.2 while the PDI of SqGem NPs lacking cholesterol was ~ 0.5. Similar or superior cytotoxicity was achieved for SqGem-based NPs compared to gemcitabine or Abraxane® when evaluated at a concentration of 10 µM. Squalenoylation of NOTA enabled in vivo monitoring of SqGem-based NP pharmacokinetics and biodistribution. Conclusion: We present a scalable technique for fabricating efficacious squalenoylated-gemcitabine nanoparticles and confirm their pharmacokinetic profile using a novel multifunctional 64Cu-SqNOTA-SqGem NP.

For the past eight decades, subcutaneous injection has been the main route used for supplementing the suboptimal insulin secretion for administering insulin as a treatment for diabetes mellitus. Although this method is effective, subcutaneous injections are painful, inconvenient and carry a high risk of infections leading to poor patient compliance. The insulin-encapsulated hepatitis E virus nanoparticle, composed of the noninfectious hepatitis E viral capsid, is expected to deliver insulin from the GI tract to the liver after ingestion. Hepatitis E virus nanoparticle could be the answer to the long search of effective and efficient means to administer insulin orally and the most preferred route of drug delivery with highest patient compliance.

Modification of membrane receptor makeup is one of the most efficient ways to control input-output signals but is usually achieved by expressing DNA or RNA-encoded proteins or by using other genome-editing methods, which can be technically challenging and produce unwanted side effects. Here we develop and validate a nanodelivery approach to transfer in vitro synthesized, functional membrane receptors into the plasma membrane of living cells. Using β2-adrenergic receptor (β2AR), a prototypical G-protein coupled receptor, as an example, we demonstrated efficient incorporation of a full-length β2AR into a variety of mammalian cells, which imparts pharmacologic control over cellular signaling and affects cellular phenotype in an ex-vivo wound-healing model. Our approach for nanodelivery of functional membrane receptors expands the current toolkit for DNA and RNA-free manipulation of cellular function. We expect this approach to be readily applicable to the synthesis and nanodelivery of other types of GPCRs and membrane receptors, opening new doors for therapeutic development at the intersection between synthetic biology and nanomedicine.

Gemcitabine (Gem) is the standard of treatment for metastatic pancreatic ductal adenocarcinoma (PDAC) and an effective anticancer drug for various solid tumors. However, clinical application of Gem chemotherapy is hampered by suboptimal delivery of the drug, mainly due to short circulation time and poor penetration of drug into the hypo-vascularized and dense fibrous stroma of PDAC. Here, we present a novel method to improve both loading and stability of Gem in temperature-sensitive liposomes (TSL). TSL have the potential to be locally activated by exposing tumor to ultrasound-mediated hyperthermia (USH) triggering release of large amounts of free drug in the tumor vasculature, which can then rapidly penetrate into the heated tumor. Gem was passively loaded into TSL composed of DPPC:DSPC:DSPE-PEG2k (80:15:5) in the presence of copper(II) gluconate and triethanolamine at final 0.12 mg-drug/mg-lipid. Formation of a CuGem complex improved drug loading and stability and reduced systemic toxicity. Mice with bilateral invasive neu deletion (NDL) tumors (4-5 mm) were treated with an i.v. administration of CuGem-TSL at 10 mg Gem/kg body weight. USH was employed to trigger the release of drug; one tumor in the bilateral tumor model was insonified with a peak ultrasound pressure of 1.1 MPa at a frequency of 1.5 MHz at 43°C for 5 min prior to and 30 min post drug injection with a variable duty cycle. We found that copper(II) gluconate possesses superior potential to solubilize Gem up to 150 mg/mL and augments passive drug loading. Upon loading Gem, formation of a complex with copper further improved drug loading and stability within TSL. By optimizing the lipid composition of the liposomal shell, we achieved high loading content of Gem at up to 12% by weight in TSL (CuGem-TSL), 4-fold greater than previously reported. Cryo electron microscopy confirmed the presence of liquid crystalline structures within CuGem-TSL, which was not observed in the absence of copper (Gem-TSL). The resulting high-content CuGem-TSL displayed a rapid release of Gem (80%) within 5 min at 42°C with only 25% release over 30 min at 37°C in the presence of serum. In vitro CuGem-TSL demonstrated equivalent toxicity to free Gem against both murine breast cancer and pancreatic cancer. One hour following intravenous administration, 75% of Gem was effectively retained in circulating TSL, whereas no drug was detected in mouse plasma with free drug administration. Three repeated administrations of CuGem-TSL combined with USH over a two-week treatment course suppressed tumor growth in NDL, model of murine breast cancer, with limited toxicity, enhancing survival when compared to treatment with free Gem alone. Additionally, in a more aggressive tumor model of murine pancreatic cancer (KrasLSL-G12D/+; Trp53LSL-R172H/+; Pdx-Cre, mT4 tumor source), CuGem-TSL with USH induced cell death and regions of apoptosis and necrosis. Citation Format: Samantha T. Tucci, Azadeh Kheirolomoom, Elizabeth S. Ingham, Lisa M. Mahakian, Sarah M. Tam, Josquin Foiret, Neil E. Hubbard, Alexander D. Borowsky, Mo Baikoghli, R. Holland Cheng, Katherine W. Ferrara. Activatable nanodelivery of high payload gemcitabine augments therapeutic efficacy in murine breast and pancreatic cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2171.

Hepatitis E Virus-like particles self-assemble in to noninfectious nanocapsids that are resistant to proteolytic/acidic mucosal delivery conditions. Previously, the nanocapsid was engineered to specifically bind and enter breast cancer cells, where successful tumor targeting was demonstrated in animal models. In the present study, the nanocapsid surface was modified with a solvent-exposed cysteine to conjugate monolayer protected gold nanoclusters (AuNC). Unlike commercially available gold nanoparticles, AuNCs monodisperse in water and are composed of a discrete number of gold atoms, forming a crystalline gold core. Au102pMBA44 (Au102) was an ideal conjugate given its small 2.5 nm size and detectability in cryoEM. Au102 was bound directly to nanocapsid surface cysteines via direct ligand exchange. In addition, Au102 was functionalized with a maleimide linker (Au102_C6MI) for maleimide-thiol conjugation to nanocapsid cysteines. The AuNC-bound nanocapsid constructs were conjugated in various conditions. We found Au102_C6MI to bind nanocapsid more efficiently, while Au102 remained more soluble over time. Nanocapsids conjugated to Au102_C6MI were imaged in cryoEM for single particle reconstruction to localize AuNC position on the nanocapsid surface. We resolved five unique high intensity volumes that formed a ring-shaped density at the 5-fold symmetry center. This finding was further supported by independent rigid modeling.