Enzyme-Responsive Nanoparticles for the Targeted Delivery of an MMP Inhibitor to Acute Myocardial Infarction.

Herein, we have developed a drug-loaded matrix metalloproteinase (MMP)-responsive micellar nanoparticle (NP) intended for minimally invasive intravenous injection during the acute phase of myocardial infarction (MI) and prolonged retention in the heart for small-molecule drug delivery. Peptide-polymer amphiphiles (PPAs) bearing a small-molecule MMP inhibitor (MMPi), PD166793, were synthesized via ring-opening metathesis polymerization (ROMP) and formulated into spherical micelles by transitioning to aqueous solution. The resulting micellar NPs underwent MMP-induced aggregation, demonstrating enzyme responsiveness. Using a rat MI model, we observed that these NPs were capable of successfully extravasating into the infarcted region of the heart where they were retained due to the active, enzyme-mediated targeting, remaining detectable after 1 week post administration without increasing macrophage recruitment. Furthermore, in vitro studies show that these NPs demonstrated successful drug release following MMP treatment and maintained drug bioactivity as evidenced by comparable MMP inhibition to free MMPi. This work establishes a targeted NP platform for delivering small-molecule therapeutics to the heart after MI, opening possibilities for myocardial infarction treatment.

Inflammation-Responsive Micellar Nanoparticles from Degradable Polyphosphoramidates for Targeted Delivery to Myocardial Infarction.

Nanoparticles that undergo a localized morphology change to target areas of inflammation have been previously developed but are limited by their lack of biodegradability. In this paper, we describe a low-ring-strain cyclic olefin monomer, 1,3-dimethyl-2-phenoxy-1,3,4,7-tetrahydro-1,3,2-diazaphosphepine 2-oxide (MePTDO), that rapidly polymerizes via ring-opening metathesis polymerization at room temperature to generate well-defined degradable polyphosphoramidates with high monomer conversion (>84%). Efficient MePTDO copolymerizations with norbornene-based monomers are demonstrated, including a norbornenyl monomer functionalized with a peptide substrate for inflammation-associated matrix metalloproteinases (MMPs). The resulting amphiphilic peptide brush copolymers self-assembled in aqueous solution to generate micellar nanoparticles (30 nm in diameter) which exhibit excellent cyto- and hemocompatibility and undergo MMP-induced assembly into micron-scale aggregates. As MMPs are upregulated in the heart postmyocardial infarction (MI), the MMP-responsive micelles were applied to target and accumulate in the infarcted heart following intravenous administration in a rat model of MI. These particles displayed a distinct biodistribution and clearance pattern in comparison to nondegradable analogues. Specifically, accumulation at the site of MI competed with elimination predominantly through the kidney rather than the liver. Together, these results suggest this as a promising new biodegradable platform for inflammation targeted delivery.

Intravascularly infused extracellular matrix as a biomaterial for targeting and treating inflamed tissues.

Decellularized extracellular matrix in the form of patches and locally injected hydrogels has long been used as therapies in animal models of disease. Here we report the safety and feasibility of an intravascularly infused extracellular matrix as a biomaterial for the repair of tissue in animal models of acute myocardial infarction, traumatic brain injury and pulmonary arterial hypertension. The biomaterial consists of decellularized, enzymatically digested and fractionated ventricular myocardium, localizes to injured tissues by binding to leaky microvasculature, and is largely degraded in about 3 d. In rats and pigs with induced acute myocardial infarction followed by intracoronary infusion of the biomaterial, we observed substantially reduced left ventricular volumes and improved wall-motion scores, as well as differential expression of genes associated with tissue repair and inflammation. Delivering pro-healing extracellular matrix by intravascular infusion post injury may provide translational advantages for the healing of inflamed tissues 'from the inside out'.

Characterization of a mGluR5 Knockout Rat Model with Hallmarks of Fragile X Syndrome.

The number of reported cases of neurodevelopmental disorders has increased significantly in the last few decades, but the etiology of these diseases remains poorly understood. There is evidence of a fundamental link between genetic abnormalities and symptoms of autism spectrum disorders (ASDs), and the most common monogenetic inheritable form of ASDs is Fragile X Syndrome (FXS). Previous studies indicate that FXS is linked to glutamate signaling regulation by the G-protein-coupled metabotropic glutamate receptor 5 (mGluR5), which has been shown to have a regulatory role in neuroinflammation. We characterized the effect of knocking out mGluR5 in an organism known to have complex cognitive functions-the rat. The heterozygous phenotype is the most clinically relevant; therefore, we performed analysis in heterozygous pups. We showed developmental abnormalities in heterozygous mGluR5 knockout rats, as well as a significant increase in chemokine (C-X-C motif) ligand 1 (CXCL) expression, a hallmark indicator of early onset inflammation. We quantified an increase in microglial density in the knockout pups and quantified morphological phenotypes representative of greater reactivity in the male vs. female and postnatal day 28 heterozygous pups compared to postnatal day 14 heterozygous pups. In response to injury, reactive microglia release matrix metalloproteases, contribute to extracellular matrix (ECM) breakdown, and are responsible for eradicating cellular and molecular debris. In our study, the changes in microglial density and reactivity correlated with abnormalities in the mRNA expression levels of ECM proteins and with the density of perineuronal nets. We saw atypical neuropsychiatric behavior in open field and elevated plus tests in heterozygous pups compared to wild-type litter and age-matched controls. These results demonstrate the pathological potential of the mGluR5 knockout in rats and further support the presence of neuroinflammatory roots in ASDs.

Targeted nanoscale therapeutics for myocardial infarction.

Nanoscale therapeutics have promise for the administration of therapeutic small molecules and biologics to the heart following myocardial infarction. Directed delivery to the infarcted region of the heart using minimally invasive routes is critical to this promise. In this review, we will discuss the advances and design considerations for two nanoscale therapeutics engineered to target the infarcted heart, nanoparticles and adeno-associated viruses.

Diffuse reflectance spectroscopy can differentiate high grade and low grade prostatic carcinoma.

Prostate tumors are graded by the revised Gleason Score (GS) which is the sum of the two predominant Gleason grades present ranging from 6-10. GS 6 cancer exclusively with Gleason grade 3 is designated as low grade (LG) and correlates with better clinical prognosis for patients. GS >7 cancer with at least one of the Gleason grades 4 and 5 is designated as HG indicate worse prognosis for patients. Current transrectal ultrasound guided prostate biopsies often fail to correctly diagnose HG prostate cancer due to sampling errors. Diffuse reflectance spectra (DRS) of biological tissue depend on tissue morphology and architecture. Thus, DRS could potentially differentiate between HG and LG prostatic carcinoma. A 15-gauge optical biopsy needle was prototyped to take prostate biopsies after measuring DRS with a laboratory fluorometer. This needle has an optical sensor that utilizes 8×100 µm optical fibers for tissue excitation and a single 200 µm central optical fiber to measure DRS. Tissue biopsy cores were obtained from 20 surgically excised prostates using this needle after measuring DRS at 5 nm intervals between 500-700 nm wavelengths. Tissue within a measurement window was histopathologically classified as either benign, LG, or HG and correlated with DRS. Partial least square analysis of DRS identified principal components (PC) as potential classifiers. Statistically significant PCs (p<;0.05) were tested for their ability to classify biopsy tissue using support vector machine and leave-one-out cross validation method. There were 29 HG and 49 LG cancers among 187 biopsy cores included in the study. Study results show 76% sensitivity, 80% specificity, 93% negative predictive value, and 50% positive predictive value for HG versus benign, and 76%, 73%, 84%, and 63%, for HG versus LG prostate tissue classification. DRS failed to diagnose 7/29 (24%) HG cancers. This study demonstrated that an optical biopsy needle guided by DRS has sufficient accuracy to differentiate HG from LG carcinoma and benign tissue. It may allow precise targeting of HG prostate cancer providing more accurate assessment of the disease and improvement in patient care.

Systematic diagnosis of prostate cancer using an optical biopsy needle adjunct with fluorescence spectroscopy.

Transrectal ultrasound guided prostate biopsies often fail to diagnose prostate cancer with 90% of cores reported as benign. Thus, it is desirable to target prostate cancer lesions while reducing the sampling of benign tissue. The concentrations of natural fluorophores in prostate tissue fluctuate with disease states. Hence, fluorescence spectroscopy could be used to quantify these fluctuations to identify prostate cancer. An optical biopsy needle with a light sensitive optical probe at the tip of the inner needle was developed to take prostate biopsies after measuring tissue fluorescence with a laboratory fluorometer. The optical probe consists of eight 100 µm fibers for tissue excitation and a single 200 µm fiber to capture fluorescence spectra. Random biopsy cores were taken from 20 surgically excised prostates after measuring fluorescence spectra of tissue between 295-550nm for several excitations between 280-350nm. Each biopsy core was histopathologically classified and correlated with corresponding spectra. Prostate biopsies were grouped into benign or malignant based on the histological findings. Out of 187 biopsy cores, 109 were benign and 78 were malignant. Partial least square analysis of tissue spectra was performed to identify diagnostically significant principal components as potential classifiers. A linear support vector machine and leave-one-out cross validation method was employed for tissue classification. Study results show 86% sensitivity, 87% specificity, 90% negative predictive value, and 83% positive predictive value for benign versus malignant prostate tissue classification. This study demonstrates potential clinical applications of fluorescence spectroscopy guided optical biopsy needle for prostate cancer diagnosis with the consequent improvement of patient care.

Anterior tumors of the prostate: diagnosis and significance.

INTRODUCTION: Prostate biopsies are usually taken from the peripheral rather than anterior region of the prostate. Consequently, tumors originating from the anterior apical region and transition zones may be under-sampled. We examined whether addition of transrectal anterior biopsy (TAB) would improve efficacy of prostate biopsies. MATERIALS AND METHODS: Simulations of TAB and sextant biopsy (SB) were performed using computer models of 86 autopsy prostates (AP) and 40 radical prostatectomy (RP) specimens. TAB was obtained bilaterally from apex, mid, and base regions by advancing the biopsy needle 5 mm-35 mm beyond the prostatic capsule. A phase I clinical trial with 114 patients was conducted to determine the performance of an extended biopsy protocol consisting of TAB, SB, and laterally-directed biopsy (LDB). RESULTS: The overall cancer detection rates of SB and TAB were 33% and 55% for AP series (p = 0.00003); 60% and 88% for RP series (p = 0.006). Alternatively, SB + bilateral apical TAB and SB + bilateral mid TAB had cancer detection rates of 45% and 42% for AP series; 80% and 78% for RP series. The extended biopsy protocol detected cancer in 33% (38/114) of patients with 29, 25, and 15 diagnosed by SB, LDB, and bilateral apical TAB, respectively. Patients diagnosed by bilateral apical TAB versus SB (p = 0.01) and LDB (p = 0.02) were statistically significant. Without bilateral apical TAB, the overall cancer detection rate decreased to 30% (34/114). CONCLUSIONS: Inclusion of bilateral TAB from apical region for first time and repeat prostate biopsies may increase diagnosis of prostate cancer. The clinical significance of these findings needs further investigations and clinical follow up.

Optimization of prostate cancer diagnosis by increasing the number of core biopsies based on gland volume.

In this prospective, non-randomized phase-I clinical trial, we comparatively studied the performance of six laterally-directed biopsies or the modified fan-shaped biopsies (MFSB), midline sextant biopsies (MB), and transition zone biopsies (TZB) and examine their prostate cancer (PCa) detection rates. A total of 114 patients received combinations of MFSB, MB, and TZB based on prostate gland volume: those ≤15 cc received 8 biopsies; those >15 cc but ≤ 50 cc received 14 biopsies; and those >50 cc received 20 biopsies. The mean prostate-specific antigen (PSA) level, Gleason score, and prostate volume were 8.0 ng/ml, 6.4, and 47 cc, respectively. PCa detection rate of the MB was 25% while the MFSB was 22%. The overall PCa detection rate was 33.3% with all biopsies. PCa and high-grade prostatic intraepithelial neoplasia (HG-PIN) detection rates decrease as the size of the prostate increases. PCa detection rates were 50.0% for volumes ≤19.9 cc and volumes of >50 cc had a detection rate of 25.8%. PSA levels of <3.0 had PCa detection rates of 15% which increased to 58% with PSA levels >9.0. In a multivariate analysis, only TZB was significant for PCa diagnosed by PSA (ß=7.4, p<0.01). Our study showed that it is important to perform both the lateral MFSB and the MB to improve overall PCa detections rates. Thus, we recommend performing MB, MFSB, and TZB based on prostate volume, as follows: 8 biopsies for ≤15 cc; 14 for those >15 cc but ≤50 cc, and 14-20 for those >50 cc.

Proliferative tumor doubling times of prostatic carcinoma.

Prostate cancer (PCa) has a variable biology ranging from latent cancer to extremely aggressive tumors. Proliferative activities of cancers may indicate their biological potential. A flow cytometric assay to calculate maximum proliferative doubling times (T(max)) of PCa in radical prostatectomy specimens after preoperative in vivo bromodeoxyuridine (BrdU) infusion is presented. Only 4/17 specimens had tumors large enough for flow cytometric analysis. The T(max) of tumors was similar and ranged from 0.6 to 3.6 months. Tumors had calculated doubling times 2- to 25-fold faster than their matched normal tissue. Variations in labeling index and T(max) were observed within a tumor as well as between different Gleason grades. The observed PSA doubling times (PSA-DT) ranged from 18.4 to 32.0 months, considerably slower than the corresponding T(max) of tumors involved. While lack of data for apoptotic rates is a limitation, apparent biological differences between latent versus aggressive PCa may be attributable to variations in apoptotic rates of these tumors rather than their cell proliferative rates.

The metabolites citrate, myo-inositol, and spermine are potential age-independent markers of prostate cancer in human expressed prostatic secretions.

OBJECTIVES: Due to specific physiological functions, prostatic tissues and fluids have unique metabolic profiles. In this study, proton nuclear magnetic resonance spectroscopy ((1)H-NMRS) is used to assess potential metabolic markers of prostate cancer (PCa) in human expressed prostatic secretions (EPS). METHODS: Metabolic profiles of EPS from 52 men with PCa and from 26 healthy controls were analyzed using quantitative (1)H-NMRS. The metabolites quantified included citrate, spermine, myo-inositol, lactate, alanine, phosphocholine, glutamine, acetate, and hydroxybutyrate. Logistic regression (LR) was used to model the risk of PCa based on metabolite concentrations while adjusting for age. RESULTS: The average age of the EPS donors with PCa was 58.0+/-7.0 years and 52.2+/-12.1 for the healthy donors. The median Gleason score for the men with PCa was 7 (range 5-9). The LR models indicated that the absolute concentrations of citrate, myo-inositol, and spermine were highly predictive of PCa and inversely related to the risk of PCa. The areas under the receiver operating characteristic curves (AUROC) for citrate, myo-inositol and spermine were 0.89, 0.87, and 0.79, respectively. At 90% sensitivity, these metabolites had specificities of 74%, 51%, and 34%, respectively. The LR analysis indicated that absolute levels of these three metabolites were independent of age. CONCLUSIONS: The results indicate that citrate, myo-inositol and spermine are potentially important markers of PCa in human EPS. Further, the absolute concentrations of these metabolites in EPS appear to be independent of age, increasing the potential utility of these markers due to elimination of age as a confounding variable.

Clinical staging of prostate cancer: a computer-simulated study of transperineal prostate biopsy.

OBJECTIVE: To identify the precise location of prostate cancer within the gland and thus possibly permit more aggressive therapy of the lesion, while potentially sparing the noncancerous gland from ablative therapy. MATERIALS AND METHODS: Three-dimensional "solid" computer models were reconstructed for 86 autopsy specimens and 20 stage T1c radical prostatectomy specimens. Transperineal biopsies were simulated for grid sizes of 5-mm (method A) and 10-mm (method B) with an 18 G, 23-mm long biopsy needle. One or two biopsies per grid point were obtained for a total of 12-108 biopsies, depending on the size of the prostate. Clinically threatening cancers were defined as having volumes of > or = 0.5 mL or Gleason sum > or = 7. RESULTS: Method A detected significantly more carcinomas than method B in both the autopsy and prostatectomy specimens (autopsy, 72 vs 51; prostatectomy, 50 vs 32, both P < 0.001). Method A also detected more clinically threatening cancers found at autopsy (38/40 vs 31/40, P = 0.008). Among autopsy patients with negative sextant biopsies whose disease was localized to one side, method A detected 72% and method B detected 29-43% (P < 0.001). CONCLUSIONS: The results of this computer simulation show that 5- and 10-mm grid biopsies detect three-quarters and a third, respectively, at autopsy, of patients with the disease localized to one side of the prostate, which may be useful when planning highly selective ablative treatments in the future.

Sulindac metabolites induce caspase- and proteasome-dependent degradation of beta-catenin protein in human colon cancer cells.

Colorectal cancer (CRC) is the second leading cause of cancer death in the USA. Accumulation of beta-catenin protein is nearly ubiquitous in colon adenomas and cancers, presumably due to mutations in the APC or beta-catenin genes that inhibit proteasome-dependent degradation of beta-catenin protein. Substantial clinical, epidemiological, and animal evidence indicate that sulindac and other non-steroidal anti-inflammatory drugs (NSAIDs) prevent the development of CRC. The mechanisms by which sulindac exerts its potent growth inhibitory effects against colon tumor cells are incompletely understood, but down-regulation of beta-catenin has been suggested as one potential mechanism. The goal of this study was to determine the mechanism of beta-catenin protein down-regulation by sulindac metabolites. Treatment of human colon cancer cell lines with apoptotic concentrations of sulindac metabolites (sulindac sulfide, sulindac sulfone) induced a dose- and time-dependent inhibition of beta-catenin protein expression. Inhibition of proteasome activity with MG-132 partially blocked the ability of sulindac sulfide and sulindac sulfone to inhibit beta-catenin protein expression. Pretreatment with the caspase inhibitor z-VAD-fmk blocked morphological signs of apoptosis as well as caspase cleavage, and also partially prevented beta-catenin degradation by sulindac metabolites. These effects occurred in cells with bi-allelic APC mutation (SW480), with wild-type APC but mono-allelic beta-catenin mutation (HCT116) and in cells that lack expression of either COX-1 or -2 (HCT15). These results indicate that loss of beta-catenin protein induced by sulindac metabolites is COX independent and at least partially due to reactivation of beta-catenin proteasome degradation and partially a result of caspase activation during the process of apoptosis.