Kit S. Lam

The effect of surface charge on in vivo biodistribution of PEG-oligocholic acid based micellar nanoparticles.

To systematically elucidate the effect of surface charge on the cellular uptake and in vivo fate of PEG-oligocholic acid based micellar nanoparticles (NPs), the distal PEG termini of monomeric PEG-oligocholic acid dendrimers (telodendrimers) are each derivatized with different number (n = 0, 1, 3 and 6) of anionic aspartic acids (negative charge) or cationic lysines (positive charge). Under aqueous condition, these telodendrimers self-assemble to form a series of micellar NPs with various surface charges, but with similar particle sizes. NPs with high surface charge, either positive or negative, were taken up more efficiently by RAW 264.7 murine macrophages after opsonization in fresh mouse serum. Mechanistic studies of cellular uptake of NPs indicated that several distinct endocytic pathways (e.g., clathrin-mediated endocytosis, caveolae-mediated endocytosis, and macropinocytosis) were involved in the cellular uptake process. After their cellular uptake, the majority of NPs were found to localize in the lysosome. Positively charged NPs exhibited dose-dependent hemolytic activities and cytotoxicities against RAW 264.7 cells proportional to the positive surface charge densities; whereas negatively charged NPs did not show obvious hemolytic and cytotoxic properties. In vivo biodistribution studies demonstrated that undesirable liver uptake was very high for highly positively or negatively charged NPs, which is likely due to active phagocytosis by macrophages (Kupffer cells) in the liver. In contrast, liver uptake was very low but tumor uptake was very high when the surface charge of NPs was slightly negative. Based on these studies, we can conclude that slightly negative charge may be introduced to the NPs surface to reduce the undesirable clearance by the reticuloendothelial system (RES) such as liver, improve the blood compatibility, thus deliver the anti-cancer drugs more efficiently to the tumor sites.

Prospective Evaluation of Cancer Clinical Trial Accrual Patterns: Identifying Potential Barriers to Enrollment

PURPOSE Well-conducted cancer clinical trials are essential for improving patient outcomes. Unfortunately, only 3% of new cancer patients participate in clinical trials. Barriers to patient accrual in cancer clinical trials must be identified and overcome to increase patient participation. MATERIALS AND METHODS We prospectively tracked factors that potentially affected patient accrual into cancer clinical trials at the University of California Davis Cancer Center. Oncologists seeing new outpatients were asked to complete questionnaires regarding patient characteristics and the physicians decision-making on patient eligibility, protocol availability, and patient opinions on participation. Statistical analysis was performed to correlate these parameters with subsequent protocol accrual. RESULTS There were 276 assessable patients. At the initial visits, physicians did not consider clinical trials in 38% (105/276) of patients principally because of a perception of protocol unavailability and poor performance status. Physicians considered 62% (171/276) of patients for participation in clinical trials. Of these, only 53% (91/171) had an appropriate protocol available for site and stage of disease. Seventy-six of 90 patients (84%) with available protocols met eligibility criteria for a particular study. Only 39 of 76 patients (51%) agreed to participate in cancer clinical trials, for an overall accrual rate of 14% (39/276). The remainder (37/76, 49%) declined trial participation despite meeting eligibility criteria. The most common reasons were a desire for other treatment (34%), distance from the cancer center (13%), patient refusal to disclose reason (11%), and insurance denial (8%). Patients with private insurance were less likely to enroll in clinical trials compared to those with government-funded insurance (OR, 0.34; P =.03; 95% CI, 0.13 to 0.9). CONCLUSION Barriers to cancer clinical trial accrual can be prospectively identified and addressed in the development and conduct of future studies, which may potentially lead to more robust clinical trials enrollment. Investigation of patient perceptions regarding the clinical trials process and the role of third party-payers is warranted.

Well-Defined, Reversible Boronate Crosslinked Nanocarriers for Targeted Drug Delivery in Response to Acidic pH Values and cis-Diols†

Stimuli-responsive nanoparticles are gaining considerable attention in the field of drug delivery due to their useful physicochemical changes in response to specific triggers, such as pH[1], temperature[2], enzymes[3] or redox conditions[4], present in certain physiological or disease microenvironment of interest. Among these nanoparticles, stimuli-responsive cross-linked micelles (SCMs) represent a versatile nanocarrier system for tumor targeting drug delivery[2c, 4-5]. For instance, SCMs exhibit superior structural stability under physiological condition compared to the non-crosslinked counterpart. As a result, these nanocarriers are able to better retain the encapsulated drug and minimize its premature release while circulating in the blood pool[2c, 4b, 5b]. The introduction of environmentally sensitive crosslinkers makes SCMs responsive to the local environment of the tumor (e.g. tumor extra-cellular pH (6.5-7.2), endosomal/lysosomal pH (4.5–6)[5b, 6], and tumor reductive intra-cellular condition[4-5]). In these instances, the payload drug is released almost exclusively in the cancerous tissue upon accumulation via the well known enhanced permeation and retention (EPR) effect[2c, 4b, 5b].

Well-defined, Reversible Disulfide Cross-linked Micelles for On-demand Paclitaxel Delivery

To minimize premature release of drugs from their carriers during circulation in the blood stream, we have recently developed reversible disulfide cross-linked micelles (DCMs) that can be triggered to release drug at the tumor site or in cancer cells. We designed and synthesized thiolated linear-dendritic polymers (telodendrimers) by introducing cysteines to the dendritic oligo-lysine backbone of our previously reported telodendrimers comprised of linear polyethylene glycol (PEG) and a dendritic cluster of cholic acids. Reversibly cross-linked micelles were then prepared by the oxidization of thiol groups to disulfide bond in the core of micelles after the self-assembly of thiolated telodendrimers. The DCMs were spherical with a uniform size of 28 nm, and were able to load paclitaxel (PTX) in the core with superior loading capacity up to 35.5% (w/w, drug/micelle). Cross-linking of the micelles within the core reduced their apparent critical micelle concentration and greatly enhanced their stability in non-reductive physiological conditions as well as severe micelle-disrupting conditions. The release of PTX from the DCMs was significantly slower than that from non-cross-linked micelles (NCMs), but can be gradually facilitated by increasing the concentration of reducing agent (glutathione) to an intracellular reductive level. The DCMs demonstrated a longer in vivo blood circulation time, less hemolytic activities, and superior toxicity profiles in nude mice, when compared to NCMs. DCMs were found to be able to preferentially accumulate at the tumor site in nude mice bearing SKOV-3 ovarian cancer xenograft. We also demonstrated that the disulfide cross-linked micellar formulation of PTX (PTX-DCMs) was more efficacious than both free drug and the non-cross-linked formulation of PTX at equivalent doses of PTX in the ovarian cancer xenograft mouse model. The anti-tumor effect of PTX-DCMs can be further enhanced by triggering the release of PTX on-demand by the administration of the FDA approved reducing agent, N-acetylcysteine, after PTX-DCMs have reached the tumor site.

A Serum Glycomics Approach to Breast Cancer Biomarkers

Because the glycosylation of proteins is known to change in tumor cells during the development of breast cancer, a glycomics approach is used here to find relevant biomarkers of breast cancer. These glycosylation changes are known to correlate with increasing tumor burden and poor prognosis. Current antibody-based immunochemical tests for cancer biomarkers of ovarian (CA125), breast (CA27.29 or CA15-3), pancreatic, gastric, colonic, and carcinoma (CA19-9) target highly glycosylated mucin proteins. However, these tests lack the specificity and sensitivity for use in early detection. This glycomics approach to find glycan biomarkers of breast cancer involves chemically cleaving oligosaccharides (glycans) from glycosylated proteins that are shed or secreted by breast cancer tumor cell lines. The resulting free glycan species are analyzed by MALDI-FT-ICR MS. Further structural analysis of the glycans can be performed in FTMS through the use of tandem mass spectrometry with infrared multiphoton dissociation. Glycan profiles were generated for each cell line and compared. These methods were then used to analyze sera obtained from a mouse model of breast cancer and a small number of serum samples obtained from human patients diagnosed with breast cancer or patients with no known history of breast cancer. In addition to the glycosylation changes detected in mice as mouse mammary tumors developed, glycosylation profiles were found to be sufficiently different to distinguish patients with cancer from those without. Although the small number of patient samples analyzed so far is inadequate to make any legitimate claims at this time, these promising but very preliminary results suggest that glycan profiles may contain distinct glycan biomarkers that may correspond to glycan “signatures of cancer.”

A self-assembling nanoparticle for paclitaxel delivery in ovarian cancer

Paclitaxel (PTX) is one of the most effective chemotherapeutic drugs for the treatment of a variety of cancers. However, it is associated with serious side effects caused by PTX itself and the Cremophor EL emulsifier. In the present study, we report the development of a well-defined amphiphilic linear-dendritic copolymer (named as telodendrimer) composed of polyethylene glycol (PEG), cholic acid (CA, a facial amphiphilic molecule) and lysine, which can form drug-loaded core/shell micelles when mixed with hydrophobic drug, such as PTX, under aqueous condition. We have used PEG(5k)-CA(8), a representive telodendrimer, to prepare paclitaxel-loaded nanoparticles (PTX-PEG(5k)-CA(8) NPs) with high loading capacity (7.3 mg PTX/mL) and a size of 20-60 nm. This novel nanoformulation of PTX was found to exhibit similar in vitro cytotoxic activity against ovarian cancer cells as the free drug (Taxol) or paclitaxel/human serum albumin nanoaggregate (Abraxane). The maximum tolerated doses (MTDs) of PTX-PEG(5k)-CA(8) NPs after single dose and five consecutive daily doses in mice were approximately 75 and 45 mg PTX/kg, respectively, which were 2.5-fold higher than those of Taxol. In both subcutaneous and orthotopic intraperitoneal murine models of ovarian cancer, PTX-PEG(5k)-CA(8) NPs achieved superior toxicity profiles and anti-tumor effects compared to Taxol and Abraxane at equivalent PTX doses, which were attributed to their preferential tumor accumulation, and deep penetration into tumor tissue, as confirmed by near infrared fluorescence (NIRF) imaging.

A smart and versatile theranostic nanomedicine platform based on nanoporphyrin

Multifunctional nanoparticles with combined diagnostic and therapeutic functions show great promise towards personalized nanomedicine. However, attaining consistently high performance of these functions in vivo in one single nano-construct remains extremely challenging. Here we demonstrate the use of one single polymer to develop a smart “all-in-one” nanoporphyrin platform that conveniently integrates a broad range of clinically relevant functions. Nanoporphyrins can be used as amplifiable multimodality nanoprobes for near-infrared fluorescence imaging (NIRFI), magnetic resonance imaging (MRI), positron emission tomography (PET) and dual modal PET-MRI. Nanoporphyrins greatly increase the imaging sensitivity for tumor detection through background suppression in blood, as well as preferential accumulation and signal amplification in tumors. Nanoporphyrins also function as multiphase nanotransducers that can efficiently convert light to heat inside tumors for photothermal-therapy (PTT), and light to singlet oxygen for photodynamic-therapy (PDT). Furthermore, nanoporphyrins act as programmable releasing nanocarriers for targeted delivery of drugs into tumors.

Directing mesenchymal stem cells to bone to augment bone formation and increase bone mass

Aging reduces the number of mesenchymal stem cells (MSCs) that can differentiate into osteoblasts in the bone marrow, which leads to impairment of osteogenesis. However, if MSCs could be directed toward osteogenic differentiation, they could be a viable therapeutic option for bone regeneration. We have developed a method to direct MSCs to the bone surface by attaching a synthetic high-affinity and specific peptidomimetic ligand (LLP2A) against integrin α4β1 on the MSC surface to a bisphosphonate (alendronate, Ale) that has a high affinity for bone. LLP2A-Ale induced MSC migration and osteogenic differentiation in vitro. A single intravenous injection of LLP2A-Ale increased trabecular bone formation and bone mass in both xenotransplantation studies and in immunocompetent mice. Additionally, LLP2A-Ale prevented trabecular bone loss after peak bone acquisition was achieved or as a result of estrogen deficiency. These results provide proof of principle that LLP2A-Ale can direct MSCs to the bone to form new bone and increase bone strength.

Congo red and thioflavin-T analogs detect Aβ oligomers

Several small molecule ligands for amyloid‐β (Aβ) fibrils deposited in brain have been developed to facilitate radiological diagnosis of Alzheimer’s disease (AD). Recently, the build‐up of Aβ oligomers (AβO) in brain has been recognized as an additional hallmark of AD and may play a more significant role in early stages. Evidence suggests that quantitative assessment of AβO would provide a more accurate index of therapeutic effect of drug trials. Therefore, there is an urgent need to develop methods for efficient identification as well as structural analysis of AβO. We found that some well established amyloid ligands, analogs of Congo red and thioflavin‐T (ThT), bind AβO with high affinity and detect AβO in vitro and in vivo. Binding studies revealed the presence of binding sites for Congo red‐ and thioflavin‐T‐analogs on AβO. Furthermore, these ligands can be used for imaging intracellular AβO in living cells and animals and as positive contrast agent for ultrastructural imaging of AβO, two applications useful for structural analysis of AβO in cells. We propose that by improving the binding affinity of current ligands, in vivo imaging of AβO is feasible by a ‘signal subtraction’ procedure. This approach may facilitate the identification of individuals with early AD.

Immunoreactivity of organic mimeotopes of the E2 component of pyruvate dehydrogenase: connecting xenobiotics with primary biliary cirrhosis.

In primary biliary cirrhosis (PBC), the major autoepitope recognized by both T and B cells is the inner lipoyl domain of the E2 component of pyruvate dehydrogenase. To address the hypothesis that PBC is induced by xenobiotic exposure, we took advantage of ab initio quantum chemistry and synthesized the inner lipoyl domain of E2 component of pyruvate dehydrogenase, replacing the lipoic acid moiety with synthetic structures designed to mimic a xenobiotically modified lipoyl hapten, and we quantitated the reactivity of these structures with sera from PBC patients. Interestingly, antimitochondrial Abs from all seropositive patients with PBC, but no controls, reacted against 3 of the 18 organic modified autoepitopes significantly better than to the native domain. By structural analysis, the features that correlated with autoantibody binding included synthetic domain peptides with a halide or methyl halide in the meta or para position containing no strong hydrogen bond accepting groups on the phenyl ring of the lysine substituents, and synthetic domain peptides with a relatively low rotation barrier about the linkage bond. Many chemicals including pharmaceuticals and household detergents have the potential to form such halogenated derivatives as metabolites. These data reflect the first time that an organic compound has been shown to serve as a mimeotope for an autoantigen and further provide evidence for a potential mechanism by which environmental organic compounds may cause PBC.