In situ T-cell transfection by anti-CD3-conjugated lipid nanoparticles leads to T-cell activation, migration, and phenotypic shift.

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 min 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.

Pre-clinical evaluation of immunoPET imaging using agonist CD40 monoclonal antibody in pancreatic tumor-bearing mice.

BACKGROUND: A novel [64Cu]Cu-NOTA-aCD40 immunoPET tracer was developed to image a CD40+ pancreatic tumor model in C57BL/6 mice and to study the biodistribution profile of the agonist CD40 (aCD40) monoclonal antibody (mAb) alone or combined with other mAbs. PROCEDURES: Copper-64 ([64Cu]Cu) labeled NOTA-aCD40 and NOTA-IgG (10 µg; 7 MBq) were injected intravenously into C57BL/6 mice with subcutaneous mT4 tumors to assess specificity 48 h post injection (p.i.) through positron emission tomography/computed tomography (PET/CT) imaging and biodistribution studies (n = 5). [64Cu]Cu-NOTA-aCD40 was injected alone or simultaneously in combination with a therapeutic mass of cold aCD40 (100 µg), aPD-1 (200 µg) and aCTLA-4 (200 µg) mAbs. A group of mice with or without tumor received the second round of injections 1 or 3 weeks apart, respectively. PET/CT imaging and biodistribution studies were performed at 48 h p.i. The organ dose for [64Cu]Cu was estimated based on biodistribution studies with 2 µg [64Cu]Cu-NOTA-aCD40 (corresponds to 5 mg patient dose) in non-tumor bearing mice. RESULTS: [64Cu]Cu-NOTA-aCD40 accumulation was 2.3- and 7.8-fold higher than [64Cu]Cu-NOTA-IgG in tumors and spleen, respectively, indicating the specificity of aCD40 mAb in a mouse pancreatic tumor model. Tumor accumulation of [64Cu]Cu-NOTA-aCD40 was 21.2 ± 7.3%ID/g at 48 h after injection. Co-injection of [64Cu]Cu-NOTA-aCD40 with cold aCD40 mAb alone or with PD-1 and CTLA-4 mAbs reduced both spleen and tumor uptake, whereas liver uptake was increased. With the second round of injections, the liver was the only organ with substantial uptake. With a 2 µg administered dose of [64Cu]Cu-NOTA-aCD40 in a dosimetry study, the liver to spleen ratio was greater compared to the 10 µg dose (2.8 vs 0.37; respectively). The human equivalent for the highest dose organ (liver) was 198 ± 28.7 µSv/MBq. CONCLUSIONS: A CD40-immunoreactive [64Cu]Cu-NOTA-aCD40 probe was developed. The ratio of spleen to liver accumulation exceeded that of the IgG isotype and was greatest with a single small, injected mass. The safety of human patient imaging with [64Cu]Cu was established based on extrapolation of the organ specificity to human imaging.

A Scalable Method for Squalenoylation and Assembly of Multifunctional 64Cu-Labeled Squalenoylated Gemcitabine Nanoparticles.

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.

Dynamic contrast enhanced MRI detects changes in vascular transport rate constants following treatment with thermally-sensitive liposomal doxorubicin.

Temperature-sensitive liposomal formulations of chemotherapeutics, such as doxorubicin, can achieve locally high drug concentrations within a tumor and tumor vasculature while maintaining low systemic toxicity. Further, doxorubicin delivery by temperature-sensitive liposomes can reliably cure local cancer in mouse models. Histological sections of treated tumors have detected red blood cell extravasation within tumors treated with temperature-sensitive doxorubicin and ultrasound hyperthermia. We hypothesize that the local release of drug into the tumor vasculature and resulting high drug concentration can alter vascular transport rate constants along with having direct tumoricidal effects. Dynamic contrast enhanced MRI (DCE-MRI) coupled with a pharmacokinetic model can detect and quantify changes in such vascular transport rate constants. Here, we set out to determine whether changes in rate constants resulting from intravascular drug release were detectable by MRI. We found that the accumulation of gadoteridol was enhanced in tumors treated with temperature-sensitive liposomal doxorubicin and ultrasound hyperthermia. While the initial uptake rate of the small molecule tracer was slower (k1=0.0478±0.011s-1 versus 0.116±0.047s-1) in treated compared to untreated tumors, the tracer was retained after treatment due to a larger reduction in the rate of clearance (k2=0.291±0.030s-1 versus 0.747±0.24s-1). While DCE-MRI assesses a combination of blood flow and permeability, ultrasound imaging of microvascular flow rate is sensitive only to changes in vascular flow rate; based on this technique, blood flow was not significantly altered 30min after treatment. In summary, DCE-MRI provides a means to detect changes that are associated with treatment by thermally-activated particles and such changes can be exploited to enhance local delivery.

Ultrasound ablation enhances drug accumulation and survival in mammary carcinoma models.

Magnetic resonance-guided focused ultrasound (MRgFUS) facilitates noninvasive image-guided conformal thermal therapy of cancer. Yet in many scenarios, the sensitive tissues surrounding the tumor constrain the margins of ablation; therefore, augmentation of MRgFUS with chemotherapy may be required to destroy remaining tumor. Here, we used 64Cu-PET-CT, MRI, autoradiography, and fluorescence imaging to track the kinetics of long-circulating liposomes in immunocompetent mammary carcinoma-bearing FVB/n and BALB/c mice. We observed a 5-fold and 50-fold enhancement of liposome and drug concentration, respectively, within MRgFUS thermal ablation-treated tumors along with dense accumulation within the surrounding tissue rim. Ultrasound-enhanced drug accumulation was rapid and durable and greatly increased total tumor drug exposure over time. In addition, we found that the small molecule gadoteridol accumulates around and within ablated tissue. We further demonstrated that dilated vasculature, loss of vascular integrity resulting in extravasation of blood cells, stromal inflammation, and loss of cell-cell adhesion and tissue architecture all contribute to the enhanced accumulation of the liposomes and small molecule probe. The locally enhanced liposome accumulation was preserved even after a multiweek protocol of doxorubicin-loaded liposomes and partial ablation. Finally, by supplementing ablation with concurrent liposomal drug therapy, a complete and durable response was obtained using protocols for which a sub-mm rim of tumor remained after ablation.

Effect of alkyl length of peptide-polymer amphiphile on cargo encapsulation stability and pharmacokinetics of 3-helix micelles.

3-Helix micelles have demonstrated excellent in vitro and in vivo stability. Previous studies showed that the unique design of the peptide-polymer conjugate based on protein tertiary structure as the headgroup is the main design factor to achieve high kinetic stability. In this contribution, using amphiphiles with different alkyl tails, namely, C16 and C18, we quantified the effect of alkyl length on the stability of 3-helix micelles to delineate the contribution of the micellar core and shell on the micelle stability. Both amphiphiles form well-defined micelles, <20 nm in size, and show good stability, which can be attributed to the headgroup design. C18-micelles exhibit slightly higher kinetic stability in the presence of serum proteins at 37 °C, where the rate constant of subunit exchange is 0.20 h(-1) for C18-micelles vs 0.22 h(-1) for C16-micelles. The diffusion constant for drug release from C18-micelles is approximately half of that for C16-micelles. The differences between the two micelles are significantly more pronounced in terms of in vivo stability and extent of tumor accumulation. C18-micelles exhibit significantly longer blood circulation time of 29.5 h, whereas C16-micelles have a circulation time of 16.1 h. The extent of tumor accumulation at 48 h after injection is ∼43% higher for C18-micelles. The present studies underscore the importance of core composition on the biological behavior of 3-helix micelles. The quantification of the effect of this key design parameter on the stability of 3-helix micelles provides important guidelines for carrier selection and use in complex environment.

A physiological perspective on the use of imaging to assess the in vivo delivery of therapeutics.

Our goal is to provide a physiological perspective on the use of imaging to optimize and monitor the accumulation of nanotherapeutics within target tissues, with an emphasis on evaluating the pharmacokinetics of organic particles. Positron emission tomography (PET), magnetic resonance imaging (MRI) and ultrasound technologies, as well as methods to label nanotherapeutic constructs, have created tremendous opportunities for preclinical optimization of therapeutics and for personalized treatments in challenging disease states. Within the methodology summarized here, the accumulation of the construct is estimated directly from the image intensity. Particle extravasation is then estimated based on classical physiological measures. Specifically, the transport of nanotherapeutics is described using the concept of apparent permeability, which is defined as the net flux of solute across a blood vessel wall per unit surface area of the blood vessel and per unit solute concentration difference across the blood vessel wall. The apparent permeability to small molecule MRI constructs is accurately shown to be far larger than that estimated for proteins such as albumin or nanoconstructs such as liposomes. Further, the quantitative measurements of vascular permeability are shown to facilitate detection of the transition from a pre-malignant to a malignant cancer and to quantify the delivery enhancement resulting from interventions such as ultrasound. While PET-based estimates facilitate quantitative comparisons of many constructs, high field MRI proves useful in the visualization of model drugs within small lesions and in the evaluation of the release and intracellular trafficking of nanoparticles and cargo.

Multimodal imaging enables early detection and characterization of changes in tumor permeability of brain metastases.

Our goal was to develop strategies to quantify the accumulation of model therapeutics in small brain metastases using multimodal imaging, in order to enhance the potential for successful treatment. Human melanoma cells were injected into the left cardiac ventricle of immunodeficient mice. Bioluminescent, MR and PET imaging were applied to evaluate the limits of detection and potential for contrast agent extravasation in small brain metastases. A pharmacokinetic model was applied to estimate vascular permeability. Bioluminescent imaging after injecting d-luciferin (molecular weight (MW) 320 D) suggested that tumor cell extravasation had already occurred at week 1, which was confirmed by histology. 7T T1w MRI at week 4 was able to detect non-leaky 100 µm sized lesions and leaky tumors with diameters down to 200 µm after contrast injection at week 5. PET imaging showed that (18)F-FLT (MW 244 Da) accumulated in the brain at week 4. Gadolinium-based MRI tracers (MW 559 Da and 2.066 kDa) extravasated after 5 weeks (tumor diameter 600 µm), and the lower MW agent cleared more rapidly from the tumor (mean apparent permeabilities 2.27 × 10(-5)cm/s versus 1.12 × 10(-5)cm/s). PET imaging further demonstrated tumor permeability to (64)Cu-BSA (MW 65.55 kDa) at week 6 (tumor diameter 700 µm). In conclusion, high field T1w MRI without contrast may improve the detection limit of small brain metastases, allowing for earlier diagnosis of patients, although the smallest lesions detected with T1w MRI were permeable only to d-luciferin and the amphipathic small molecule (18)F-FLT. Different-sized MR and PET contrast agents demonstrated the gradual increase in leakiness of the blood tumor barrier during metastatic progression, which could guide clinicians in choosing tailored treatment strategies.

Evaluation of doxorubicin-loaded 3-helix micelles as nanocarriers.

Designing stable drug nanocarriers, 10-30 nm in size, would have significant impact on their transport in circulation, tumor penetration, and therapeutic efficacy. In the present study, biological properties of 3-helix micelles loaded with 8 wt % doxorubicin (DOX), ~15 nm in size, were characterized to validate their potential as a nanocarrier platform. DOX-loaded micelles exhibited high stability in terms of size and drug retention in concentrated protein environments similar to conditions after intravenous injections. DOX-loaded micelles were cytotoxic to PPC-1 and 4T1 cancer cells at levels comparable to free DOX. 3-Helix micelles can be disassembled by proteolytic degradation of peptide shell to enable drug release and clearance to minimize long-term accumulation. Local administration to normal rat striatum by convection enhanced delivery (CED) showed greater extent of drug distribution and reduced toxicity relative to free drug. Intravenous administration of DOX-loaded 3-helix micelles demonstrated improved tumor half-life and reduced toxicity to healthy tissues in comparison to free DOX. In vivo delivery of DOX-loaded 3-helix micelles through two different routes clearly indicates the potential of 3-helix micelles as safe and effective nanocarriers for cancer therapeutics.

Long-circulating 15 nm micelles based on amphiphilic 3-helix peptide-PEG conjugates.

Generating stable, multifunctional organic nanocarriers will have a significant impact on drug formulation. However, it remains a significant challenge to generate organic nanocarriers with a long circulation half-life, effective tumor penetration, and efficient clearance of metabolites. We have advanced this goal by designing a new family of amphiphiles based on coiled-coil 3-helix bundle forming peptide-poly(ethylene glycol) conjugates. The amphiphiles self-assemble into monodisperse micellar nanoparticles, 15 nm in diameter. Using the 3-helix micelles, a drug loading of ∼8 wt % was obtained using doxorubicin and the micelles showed minimal cargo leakage after 12 h of incubation with serum proteins at 37 °C. In vivo pharmacokinetics studies using positron emission tomography showed a circulation half-life of 29.5 h and minimal accumulation in the liver and spleen. The demonstrated strategy, by incorporating unique protein tertiary structure in the headgroup of an amphiphile, opens new avenues to generate organic nanoparticles with tunable stability, ligand clustering, and controlled disassembly to meet current demands in nanomedicine.

Specific penetration and accumulation of a homing peptide within atherosclerotic plaques of apolipoprotein E-deficient mice.

The ability to selectively deliver compounds into atherosclerotic plaques would greatly benefit the detection and treatment of atherosclerotic disease. We describe such a delivery system based on a 9-amino acid cyclic peptide, LyP-1. LyP-1 was originally identified as a tumor-homing peptide that specifically recognizes tumor cells, tumor lymphatics, and tumor-associated macrophages. As the receptor for LyP-1, p32, is expressed in atherosclerotic plaques, we tested the ability of LyP-1 to home to plaques. Fluorescein-labeled LyP-1 was intravenously injected into apolipoprotein E (ApoE)-null mice that had been maintained on a high-fat diet to induce atherosclerosis. LyP-1 accumulated in the plaque interior, predominantly in macrophages. More than 60% of cells released from plaques were positive for LyP-1 fluorescence. Another plaque-homing peptide, CREKA, which binds to fibrin-fibronectin clots and accumulates at the surface of plaques, yielded fewer positive cells. Tissues that did not contain plaque yielded only traces of LyP-1(+) cells. LyP-1 was capable of delivering intravenously injected nanoparticles to plaques; we observed abundant accumulation of LyP-1-coated superparamagnetic iron oxide nanoparticles in the plaque interior, whereas CREKA-nanoworms remained at the surface of the plaques. Intravenous injection of 4-[(18)F]fluorobenzoic acid ([(18)F]FBA)-conjugated LyP-1 showed a four- to sixfold increase in peak PET activity in aortas containing plaques (0.31% ID/g) compared with aortas from normal mice injected with [(18)F]FBA-LyP-1(0.08% ID/g, P < 0.01) or aortas from atherosclerotic ApoE mice injected with [(18)F]FBA-labeled control peptide (0.05% ID/g, P < 0.001). These results indicate that LyP-1 is a promising agent for the targeting of atherosclerotic lesions.

Longitudinal investigation of permeability and distribution of macromolecules in mouse malignant transformation using PET.

PURPOSE: We apply positron emission tomography (PET) to elucidate changes in nanocarrier extravasation during the transition from premalignant to malignant cancer, providing insight into the use of imaging to characterize early cancerous lesions and the utility of nanoparticles in early disease. EXPERIMENTAL DESIGN: Albumin and liposomes were labeled with (64)Cu (half-life 12.7 hours), and longitudinal PET and CT imaging studies were conducted in a mouse model of ductal carcinoma in situ. A pharmacokinetic model was applied to estimate the tumor vascular volume and permeability. RESULTS: From early time points characterized by disseminated hyperproliferation, the enhanced vascular permeability facilitated lesion detection. During disease progression, the vascular volume fraction increased 1.6-fold and the apparent vascular permeability to albumin and liposomes increased ∼2.5-fold to 6.6 × 10(-8) and 1.3 × 10(-8) cm/s, respectively, with the accumulation of albumin increasing earlier in the disease process. In the malignant tumor, both tracers reached similar mean intratumoral concentrations of ∼6% ID/cc but the distribution of liposomes was more heterogeneous, ranging from 1% to 18% ID/cc compared with 1% to 9% ID/cc for albumin. The tumor-to-muscle ratio was 17.9 ± 8.1 and 7.1 ± 0.5 for liposomes and albumin, respectively, indicating a more specific delivery of liposomes than with albumin. CONCLUSIONS: PET imaging of radiolabeled particles, validated by confocal imaging and histology, detected the transition from premalignant to malignant lesions and effectively quantified the associated changes in vascular permeability.

Imaging nanoparticle stability and activation in vivo.

While liposomes and nanoparticles have been the subject of intense research for more than 40 years, few particles have been translated into clinical practice. Advantages of these particles include the potential to overcome the cardiac, renal or neural toxicity of systemic chemotherapy, the opportunities for multivalent targeting, the gradual yet significant accumulation within tumors due to leaky blood vessels and the myriad of new approaches to locally alter the properties of the particle in the region of interest. Given the complexity of the design and co-optimization of the surface architecture, shell formulation and drug loading, methods to image the pharmacokinetics of nanoparticles in living systems are an essential part of an efficient research methodology. Here, we describe our efforts to label the shell and drug core of lipid-shelled particles with a goal of facilitating translation of activatable particles.

Dynamic imaging of arginine-rich heart-targeted vehicles in a mouse model.

Efficacious delivery of drugs and genes to the heart is an important goal. Here, a radiolabeled peptide-targeted liposome was engineered to bind to the heart, and the biodistribution and pharmacokinetics were determined by dynamic positron emission tomography in the FVB mouse. Efficient targeting occurred only with an exposed ligand and a dense concentration of peptide (6000 peptides/particles). Liposomes targeted with CRPPR or other arginine-rich peptides with an exposed guanidine moiety bound within 100 s after intravenous injection and remained stably bound. With CRPPR-targeted particles, the radioisotope density in the heart averaged 44 +/- 9% injected dose/gram of tissue, more than 30-fold higher than in skeletal muscle. The rapid and efficient targeting of these particles can be exploited in drug and gene delivery systems and with dynamic positron emission tomography provides a model system to optimize targeting of engineered particles.