Targeted Imaging Agent to HSP70 Induced In Vivo.

Heat shock protein expression can be induced by heat shock making it possible to artificially modulate their levels noninvasively in vivo in a spatially and temporally controlled manner. Here, we report the use of the major heat shock protein 70 (HSP70) as an inducible target by using the small molecule deoxyspergualin (DSG) conjugated to the near-infrared fluorophore (Cy5.5). We demonstrate that heat induction in the form of localized hyperthermia of normal tissue in living mice results in sufficient HSP70 overexpression for detection with DSG-Cy5.5 conjugate. This effect is dependent on total energy delivered and reaches maximum fluorescence signal in 6 to 8 hours post heat induction and declines over a period of up to 24 hours. These results suggest that DSG-Cy5.5 agent accumulates in tissue with elevated HSP70 by heat.

A naturally occurring single amino acid replacement in multiple gene regulator of group A Streptococcus significantly increases virulence.

Single-nucleotide polymorphisms (SNPs) are the most common source of genetic variation within a species; however, few investigations demonstrate how naturally occurring SNPs may increase strain virulence. We recently used group A Streptococcus as a model pathogen to study bacteria strain genotype-patient disease phenotype relationships. Whole-genome sequencing of approximately 800 serotype M59 group A Streptococcus strains, recovered during an outbreak of severe invasive infections across North America, identified a disproportionate number of SNPs in the gene encoding multiple gene regulator of group A Streptococcus (mga). Herein, we report results of studies designed to test the hypothesis that the most commonly occurring SNP, encoding a replacement of arginine for histidine at codon 201 of Mga (H201R), significantly increases virulence. Whole transcriptome analysis revealed that the H201R replacement significantly increased expression of mga and 54 other genes, including many proven virulence factors. Compared to the wild-type strain, a H201R isogenic mutant strain caused significantly larger skin lesions in mice. Serial quantitative bacterial culture and noninvasive magnetic resonance imaging also demonstrated that the isogenic H201R strain was significantly more virulent in a nonhuman primate model of joint infection. These findings show that the H201R replacement in Mga increases the virulence of M59 group A Streptococcus and provide new insight to how a naturally occurring SNP in bacteria contributes to human disease phenotypes.

Non-lethal heat treatment of cells results in reduction of tumor initiation and metastatic potential.

Non-lethal hyperthermia is used clinically as adjuvant treatment to radiation, with mixed results. Denaturation of protein during hyperthermia treatment is expected to synergize with radiation damage to cause cell cycle arrest and apoptosis. Alternatively, hyperthermia is known to cause tissue level changes in blood flow, increasing the oxygenation and radiosensitivity of often hypoxic tumors. In this study, we elucidate a third possibility, that hyperthermia alters cellular adhesion and mechanotransduction, with particular impact on the cancer stem cell population. We demonstrate that cell heating results in a robust but temporary loss of cancer cell aggressiveness and metastatic potential in mouse models. In vitro, this heating results in a temporary loss in cell mobility, adhesion, and proliferation. Our hypothesis is that the loss of cellular adhesion results in suppression of cancer stem cells and loss of tumor virulence and metastatic potential. Our study suggests that the metastatic potential of cancer is particularly reduced by the effects of heat on cellular adhesion and mechanotransduction. If true, this could help explain both the successes and failures of clinical hyperthermia, and suggest ways to target treatments to those who would most benefit.

Specific binding of modified ZD6474 (Vandetanib) monomer and its dimer with VEGF receptor-2.

Tumor angiogenesis is an important component of cancer biology driven in part by the hypothesis that tumor vessel growth is a necessary requirement for tumor growth. Angiogenesis does not only depend on endothelial cell invasion and proliferation, it also requires pericyte coverage of vascular sprouts for vessel stabilization. These processes are coordinated by vascular endothelial growth factor (VEGF). Vandetanib, also known as ZD6474, is an orally bioavailable small molecule tyrosine kinase inhibitor of multiple growth factors that is an antagonist of the vascular endothelial growth factor receptor-2 (VEGFR-2). ZD6474 was purchased and modified to add a fluorescent dye (6-FAM). A linker was used to couple the ZD6474 monomer to create dimers, and similarly linked to FAM fluorescent dye. The two compounds were compared using human endothelial cell (HUVECs) and the cancer cell lines U-87-MG and MDA-MB-231. We compared cellular uptake and binding specificity, as well as effects on cellular viability and angiogenesis in a series of in vitro and in vivo studies. ZD6474 dimer demonstrated improved uptake in HUVECs and other VEGFR expressing cells over ZD6474 monomer in vitro. Therapeutic effects were mixed, with in vitro studies showing the dimer having the same or weaker effects compared to the monomer, while an in vivo study using pseudotumors (matrigel plug assay) seemed to indicate stronger effects could be obtained from the dimer. Finally, in biodistribution study in a xenograft tumor model, the dimer accumulated 20× greater concentration in the tumor than in the liver, spleen, or kidneys, and also 20× greater than the accumulation of the monomer or the dye alone, all at 24 h following compound injection. This study provides a rationale for the evaluation of dimeric ZD6474 as a potent imaging agent of angiogenic activity in vivo.

MRI-based prediction of pulsed high-intensity focused ultrasound effect on tissue transport in rabbit muscle.

PURPOSE: To design an algorithm for optimizing pulsed high intensity focused ultrasound (p-HIFU) treatment parameters to maximize tissue transport while minimizing thermal necrosis based on MR image guidance. MATERIALS AND METHODS: P-HIFU power, duty cycle, and treatment duration were varied to generate different levels of thermal and mechanical deposition in rabbit muscle. Changes in T2-weighted and T1 contrast-enhanced (CE) signal were assessed immediately following treatment and at 24 h. Transport parameters were extracted by means of T1-weighted dynamic contrast-enhanced MRI (DCE-MRI) technique at 0 and 24-h time points. RESULTS: Successful p-HIFU treatment was indicated by focal hyperintensity on the T2-weighted image immediately post-treatment, suggesting increased fluid (edema), with little intensity change in CE image. After 24 h, the affected region expanded along the muscle fiber accompanied by clear hyperintensity in CE image (contrast uptake). Quantitative DCE-MRI analysis revealed statistically significant increases in both leakage rate and extracellular space, accompanied by a decrease in clearance rate. CONCLUSION: Successful p-HIFU treatment was mainly correlated to tissue heating. Tissue transport properties following treatment success would result in improved contact between drug and targets in both time and space. MRI is the key to controlling treatment by means of thermometry and also monitoring efficacy by means of T2-weighted imaging.

Multivalency of non-peptide integrin αVß3 antagonist slows tumor growth.

Multivalency is a powerful strategy for achieving high-affinity molecular binding of compounds to increase their therapeutic potency or imaging potential. In our study, multivalent non-peptide integrin αvß3 antagonists (IA) were designed for antitumor therapy. Docking and molecular dynamics were employed to explore the binding modes of IA monomer, dimer, and trimer. In silico, one IA unit binds tightly in the active site with similar pose to native ligand RGD and other parts of dimer and trimer contribute extra binding affinities by interacting with residues in vicinity of the original site. In vitro studies demonstrated that increasing valency results in increasing antiproliferative and antiorganizational effects against endothelial cells (HUVECs), and a much weaker effect on melanoma B16F10 cells. The antitumor efficacies of the IA multivalent compounds were evaluated in subcutaneous B16F10 melanoma tumor-bearing mice. At 30 mg/kg dose, the mean masses of tumors harvested 18 days after inoculation were significantly reduced (p<10(-7)) by 36±9%, 49±8%, and 71±7% for the IA monomer, dimer, and trimer groups, relative to control. The importance of multivalency was demonstrated to be highly significant beyond the additive effect of the extra pharmacological sites (p=0.00011). These results suggest that the major target of these anti-αvß3 compounds is the neovasculature rather than the cancer cells, and the success of a multivalent strategy depends on the details of the components and linker. This is the first integrin αvß3 multivalent ligand showing clear enhancement in antitumor effectiveness.

Efficient electroporation of liposomes doped with pore stabilizing nisin.

CONTEXT: Liposomes have a long history as passive and active drug carriers. Recently, a few methods have been realized to control the release from liposomes, including heating, ultrasound and laser. OBJECTIVE: We report on a new approach to drive release from liposomes using electric fields. MATERIALS AND METHODS: Liposomes were manufactured containing a high concentration of (quenched) 5-6 carboxyfluorescein dye. Nisin, a well-known amphiphilic peptide lantibiotic that works by stabilizing pores formed in cell membranes, was mixed in solution inside or outside the liposomes. The liposomes were then electroporated using a range of voltages, and assayed for increases in fluorescence due to release of dye. Release was measured against positive and negative controls, with positive control release driven by a strong detergent. RESULTS: Our results demonstrate that the addition of nisin significantly reduces the electric field required to release the contents of liposomes, from 2000 V/m to approximately 200 V/m. This result proves that, in principle, electroporation (EP) of liposomes doped with small amounts of amphiphilic pore stabilizing peptides may be a practical means to drive release of liposomal contents in vivo. CONCLUSION: Drug delivery from liposomes doped with amphiphilic peptides using EP is feasible. This technique could be developed into a potent adjuvant to tumor ablation using irreversible EP.

Estimation of thermal dose from MR thermometry during application of nonablative pulsed high intensity focused ultrasound.

PURPOSE: To evaluate whether MR thermometry is sufficiently fast, accurate, and spatially resolved for monitoring the thermal safety of nonablative pulsed high intensity ultrasound (pHIFU) treatments. MATERIALS AND METHODS: A combination of real MR thermometry data and modeling was used to analyze the effects of temporal and spatial averaging as well as noise on the peak temperatures and thermal doses that would be measured by MR thermometry. RESULTS: MR thermometry systematically underestimates the temperature and thermal doses during pHIFU treatment. Small underestimates of peak temperature can lead to large underestimates of thermal dose. Spatial averaging errors are small for ratios of pixel dimension to heating zone radius less than 0.25, which may be achieved by reducing the voxel size or steering the acoustic beam. Thermal dose might also be underestimated for very short, high power pulses due to temporal averaging. A simple correction factor based on the applied power and duty cycle may be applied to determine the upper bound of this effect. CONCLUSION: The temperature and thermal dose measured using MR thermometry during pulsed HIFU treatment is probably sufficient in most instances. Simple corrections may be used to calculate an upper bound where this is a critical factor.

Partially polymerized liposomes: stable against leakage yet capable of instantaneous release for remote controlled drug delivery.

A critical issue for current liposomal carriers in clinical applications is their leakage of the encapsulated drugs that are cytotoxic to non-target tissues. We have developed partially polymerized liposomes composed of polydiacetylene lipids and saturated lipids. Cross-linking of the diacetylene lipids prevents the drug leakage even at 40 °C for days. These inactivated drug carriers are non-cytotoxic. Significantly, more than 70% of the encapsulated drug can be instantaneously released by a laser that matches the plasmon resonance of the tethered gold nanoparticles on the liposomes, and the therapeutic effect was observed in cancer cells. The remote activation feature of this novel drug delivery system allows for precise temporal and spatial control of drug release.

Augmentation of targeted delivery with pulsed high intensity focused ultrasound.

This paper reviews the enhanced delivery of genes, drugs and therapeutics using ultrasound. It begins with a general overview of the field and the various techniques associated with it, including sonophoresis, hyperthermia (with ultrasound), sonoporation, and microbubble assisted transvascular and targeted delivery. Particular attention is then paid to pulsed high intensity focused ultrasound drug delivery without the use of ultrasound contrast agents. Feasibility and mechanistic studies of this technique are described in some detail. Conclusions are then drawn regarding possible mechanisms of this treatment, and to contrast with the better known treatments relying on injection of ultrasound contrast agents.

Tumor ablation using low-intensity ultrasound and sound excitable drug.

The cell membrane is a semi-fluid container that defines the boundary of cells, and provides an enclosed environment for vital biological processes. A sound excitable drug (SED) that is non-cytotoxic to cells is developed to disrupt the plasma membrane under gentle ultrasound insonation, 1MHz, 1W/cm2. The frequency and power density of insonation are within the physical therapy and medical imaging windows; thus the applied ultrasound is safe and not harmful to tissues. The insertion of SEDs into the plasma membrane is not toxic to cells; however, the intruding SEDs weaken the membrane's integrity. Under insonation, the ultrasound energy destabilized the SED disrupted membranes, resulting in membrane rupture and eventual cell death. In a xenograft breast tumor model, the SED alone or the ultrasound alone caused little adverse effects to tumor tissue, while the combined treatment triggered necrosis with a brief local insonation of 3min. The described sono-membrane rupture therapy could be a safe alternative to the currently used high-energy tissue ablation technology, which uses X-rays, gamma rays, electron beams, protons, or high-intensity focused ultrasound.

Intraoperative diagnostics and elimination of residual microtumours with plasmonic nanobubbles.

Failure of cancer surgery to intraoperatively detect and eliminate microscopic residual disease (MRD) causes lethal recurrence and metastases, and the removal of important normal tissues causes excessive morbidity. Here, we show that a plasmonic nanobubble (PNB), a non-stationary laser pulse-activated nanoevent, intraoperatively detects and eliminates MRD in the surgical bed. PNBs were generated in vivo in head and neck cancer cells by systemically targeting tumours with gold colloids and locally applying near-infrared, low-energy short laser pulses, and were simultaneously detected with an acoustic probe. In mouse models, between 3 and 30 residual cancer cells and MRD (undetectable with current methods) were non-invasively detected up to 4 mm deep in the surgical bed within 1 ms. In resectable MRD, PNB-guided surgery prevented local recurrence and delivered 100% tumour-free survival. In unresectable MRD, PNB nanosurgery improved survival twofold compared with standard surgery. Our results show that PNB-guided surgery and nanosurgery can rapidly and precisely detect and remove MRD in simple intraoperative procedures.

Polymeric micelles and nanoemulsions as tumor-targeted drug carriers: Insight through intravital imaging.

Intravital imaging of nanoparticle extravasation and tumor accumulation has revealed, for the first time, detailed features of carrier and drug behavior in circulation and tissue that suggest new directions for optimization of drug nanocarriers. Using intravital fluorescent microscopy, the extent of the extravasation, diffusion in the tissue, internalization by tissue cells, and uptake by the RES system were studied for polymeric micelles, nanoemulsions, and nanoemulsion-encapsulated drug. Discrimination of vascular and tissue compartments in the processes of micelle and nanodroplet extravasation and tissue accumulation was possible. A simple 1-D continuum model was suggested that allowed discriminating between various kinetic regimes of nanocarrier (or released drug) internalization in tumors of various sizes and cell density. The extravasation and tumor cell internalization occurred much faster for polymeric micelles than for nanoemulsion droplets. Fast micelle internalization resulted in the formation of a perivascular fluorescent coating around blood vessels. A new mechanism of micelle extravasation and internalization was suggested, based on the fast extravasation and internalization rates of copolymer unimers while maintaining micelle/unimer equilibrium in the circulation. The data suggested that to be therapeutically effective, nanoparticles with high internalization rate should manifest fast diffusion in the tumor tissue in order to avoid generation of concentration gradients that induce drug resistance. However an extra-fast diffusion should be avoided as it may result in the flow of extravasated nanoparticles from the tumor to normal organs, which would compromise targeting efficiency. The extravasation kinetics were different for nanodroplets and nanodroplet-encapsulated drug F-PTX suggesting a premature release of some fraction of the drug from the carrier. In conclusion, the development of an "ideal" drug carrier should involve the optimization of both drug retention and carrier diffusion parameters.

Safety and efficacy of quadrapeutics versus chemoradiation in head and neck carcinoma xenograft model.

Chemoradiation is the strongest anti-tumor therapy but in resistant unresectable cancers it often lacks safety and efficacy. We compared our recently developed cell-level combination approach, quadrapeutics, to chemoradiation therapy to establish pre-clinical data for its biodistribution, safety and efficacy in head and neck squamous cell carcinoma (HNSCC), as a clinically challenging aggressive and resistant cancer. In vitro and in vivo models of four carcinomas were treated with standard chemoradiation and quadrapeutics using identical drug and radiation doses. We applied liposomal cisplatin or doxorubicin, colloidal gold, near-infrared laser pulses and radiation, all at low safe doses. The final evaluation used a xenograft model of HNSCC. Quadrapeutics enhanced standard chemoradiation in vitro by reducing head and neck cancer cell proliferation by 1000-fold, inhibiting tumor growth in vivo by 34-fold and improving animal survival by 5-fold, and reducing the side effects to a negligible level. In quadrapeutics, we observed an "inversion" of the drug efficacy of two standard drugs: doxorubicin, a low efficacy drug for the cancers studied, was two times more efficient than cisplatin, the first choice drug in clinic for HNSCC. The radical therapeutic gain of quadrapeutics resulted from the intracellular synergy of the four components employed which we administered in a specific sequence, while the reduction in the toxicity was due to the low doses of all four components. The biodistribution, safety and efficacy data for quadrapeutics in HNSCC ensure its high translational potential and justify the possibility of clinical trials.

HSP70 promoter-driven activation of gene expression for immunotherapy using gold nanorods and near infrared light.

Modulation of the cytokine milieu is one approach for vaccine development. However, therapy with pro-inflammatory cytokines, such as IL-12, is limited in practice due to adverse systemic effects. Spatially-restricted gene expression circumvents this problem by enabling localized amplification. Intracellular co-delivery of gold nanorods (AuNR) and a heat shock protein 70 (HSP70) promoter-driven expression vector enables gene expression in response to near infrared (NIR) light. AuNRs absorb the light, convert it into heat and thereby stimulate photothermal expression of the cytokine. As proof-of-concept, human HeLa and murine B16 cancer cells were transfected with a HSP70-Enhanced Green Fluorescent Protein (EGFP) plasmid and polyethylenimine (PEI)-conjugated AuNRs. Exposure to either 42 °C heat-shock or NIR light induced significant expression of the reporter gene. In vivo NIR driven expression of the reporter gene was confirmed at 6 and 24 h in mice bearing B16 melanoma tumors using in vivo imaging and flow-cytometric analysis. Overall, we demonstrate a novel opportunity for site-directed, heat-inducible expression of a gene based upon the NIR-absorbing properties of AuNRs and a HSP70 promoter-driven expression vector.

Phase-shift, stimuli-responsive drug carriers for targeted delivery.

The intersection of particles and directed energy is a rich source of novel and useful technology that is only recently being realized for medicine. One of the most promising applications is directed drug delivery. This review focuses on phase-shift nanoparticles (that is, particles of submicron size) as well as micron-scale particles whose action depends on an external-energy triggered, first-order phase shift from a liquid to gas state of either the particle itself or of the surrounding medium. These particles have tremendous potential for actively disrupting their environment for altering transport properties and unloading drugs. This review covers in detail ultrasound and laser-activated phase-shift nano- and micro-particles and their use in drug delivery. Phase-shift based drug-delivery mechanisms and competing technologies are discussed.

Cancer treatment using an optically inert Rose Bengal derivative combined with pulsed focused ultrasound.

Pulsed high intensity focused ultrasound (HIFU) has been combined with a photo-insensitive Rose Bengal derivative (RB2) to provide a synergistic cytotoxicity requiring the presence of both ultrasonic cavitation and drug. In vitro tests have shown that a short treatment (less than 30 s) of pulsed HIFU with peak negative pressure >7 MPa (~27 W acoustic power at 1.4 MHz) destroys >95% of breast cancer cells MDA-MB-231 in suspension with >10 µM of the compound. Neither the pulsed HIFU nor the RB2 compound was found to have any significant impact on the viability of the cells when used alone. Introducing an antioxidant (N-acetylcysteine) reduced the effectiveness of the treatment. In vivo tests using these same cells growing as a xenograft in nu/nu mice were also done. An ultrasound contrast agent (Optison) and lower frequency (1.0 MHz) was used to help initiate cavitation at the tumor site. We were able to demonstrate tumor regression with cavitation alone, however, addition of RB2 compound injected i.v. yielded a substantial synergistic improvement.

Pulsed high intensity focused ultrasound mediated nanoparticle delivery: mechanisms and efficacy in murine muscle.

High intensity focused ultrasound (HIFU) is generally thought to interact with biological tissues in two ways: hyperthermia (heat) and acoustic cavitation. Pulsed mode HIFU has recently been demonstrated to increase the efficacy of a variety of drug therapies. Generally, it is presumed that the treatment acts to temporarily increase the permeability of the tissue to the therapeutic agent, however, the precise mechanism remains in dispute. In this article, we present evidence precluding hyperthermia as a principal mechanism for enhancing delivery, using a quantitative analysis of systemically administered fluorescent nanoparticles delivered to muscle in the calves of mice. Comparisons were carried out on the degree of enhancement between an equivalent heat treatment, delivered without ultrasound, and that of the pulsed-HIFU itself. In the murine calf muscle, Pulsed-HIFU treatment resulted in a significant increase in distribution of 200 nm particles (p < 0.016, n = 6), while the equivalent thermal dose showed no significant increase. Additional studies using this tissue/agent model also demonstrated that the pulsed HIFU enhancing effects persist for more than 24 h, which is longer than that of hyperthermia and acoustic cavitation, and offers the possibility of a novel third mechanism for mediating delivery.

Application of a nonlinear boundary condition model to adhesion interphase damage and failure.

In an earlier paper [J. Sadler, B. O'Neill, and R. Maev, J. Acoust. Soc. Am. 118, 51-59 (2005)], a set of generalized boundary conditions were proposed, based on a thin layer (thickness << wavelength) model of the acoustic interface. In this paper, the model is extended to cover the more pathological nonlinearity of the adhesion interphase-that is, the critically important thin layer where bonds are formed between adhesive and substrate. First, the boundary conditions are shown to be sufficiently general to cope with all manner of interphase nonlinearity, including unilateral cases such as clapping or slipping. To maintain this generality, an analytic time domain solution is proposed based on expansion in terms of the layer thickness rather than the conventional expansion in terms of harmonics. Finally, the boundary conditions are applied to an interphase failure model based upon basic continuum damage mechanics principles. It is proposed that such a model, which can predict the evolution of the interphase damage under stressful conditions, may allow a proper prediction of the ultimate adhesion strength based on nonlinear parameters measured nondestructively with ultrasound.