Timothy M. Fan

Investigating the optimal size of anticancer nanomedicine

Significance Understanding the interdependency of physiochemical properties of nanomedicine (NM) in correlation to its biological response and function is crucial for additional development of anticancer NM. Here, we prepared monodisperse drug–silica nanoconjugates in three distinct sizes (20, 50, and 200 nm) with other physiochemical properties controlled to be identical to investigate size-dependent biodistribution, tumor tissue penetration and clearance, and anticancer efficacy in various tumor models. We also developed a mathematical model of the spatiotemporal distribution of NM within a tumor to gain insight into the size-dependent interaction with tumor. Our studies show clear evidence that there is an optimal size of anticancer NM and that NM with the optimal size has the highest tumor retention integrated over time. Nanomedicines (NMs) offer new solutions for cancer diagnosis and therapy. However, extension of progression-free interval and overall survival time achieved by Food and Drug Administration-approved NMs remain modest. To develop next generation NMs to achieve superior anticancer activities, it is crucial to investigate and understand the correlation between the physicochemical properties of NMs (particle size in particular) and their interactions with biological systems to establish criteria for NM optimization. Here, we systematically evaluated the size-dependent biological profiles of three monodisperse drug–silica nanoconjugates (NCs; 20, 50, and 200 nm) through both experiments and mathematical modeling and aimed to identify the optimal size for the most effective anticancer drug delivery. Among the three NCs investigated, the 50-nm NC shows the highest tumor tissue retention integrated over time, which is the collective outcome of deep tumor tissue penetration and efficient cancer cell internalization as well as slow tumor clearance, and thus, the highest efficacy against both primary and metastatic tumors in vivo.

Synthesis and Biological Response of Size-Specific, Monodisperse Drug-Silica Nanoconjugates

Drug-containing nanoparticles (NPs) with monodisperse, controlled particle sizes are highly desirable for drug delivery. Accumulating evidence suggests that NPs with sizes less than 50 nm demonstrate superior performance in vitro and in vivo. However, it is difficult to fabricate monodisperse, drug-containing NPs with discrete sizes required for studying and characterizing existing relationships among particle size, biologic processing, and therapeutic functionality. Here, we report a scalable process of fabricating drug-silica conjugated nanoparticles, termed drug-silica nanoconjugates (drug-NCs), which possess monodisperse size distributions and desirable particle sizes as small as 20 nm. We find that 20 nm NCs are superior to their 50 and 200 nm NC analogues by 2-5- and 10-20-fold, respectively, with regard to tumor accumulation and penetration and cellular internalization. These fundamental findings underscore the importance and necessity of further miniaturizing nanomedicine size for optimized drug delivery applications.

The formulation of aptamer-coated paclitaxel―polylactide nanoconjugates and their targeting to cancer cells

Paclitaxel-polylactide (Ptxl-PLA) conjugate nanoparticles, termed as nanoconjugates (NCs), were prepared through Ptxl/(BDI)ZnN(TMS)(2) (BDI = 2-((2,6-diisopropylphenyl)-amido)-4-((2,6-diisopropylphenyl)-imino)-2-pentene)-mediated controlled polymerization of lactide (LA) followed by nanoprecipitation. Nanoprecipitation of Ptxl-PLA resulted in sub-100 nm NCs with monomodal particle distributions and low polydispersities. The sizes of Ptxl-PLA NCs could be precisely controlled by using appropriate water-miscible solvents and by controlling the concentration of Ptxl-PLA during nanoprecipitation. Co-precipitation of a mixture of PLA-PEG-PLA (PLA = 14 kDa; PEG = 5 kDa) and Ptxl-PLA in PBS resulted in NCs that could stay non-aggregated in PBS for an extended period of time. To develop solid formulations of NCs, we evaluated a series of lyoprotectants, aiming to identify candidates that could effectively reduce or eliminate NC aggregation during lyophilization. Albumin was found to be an excellent lyoprotectant for the preparation of NCs in solid form, allowing lyophilized NCs to be readily dispersed in PBS without noticeable aggregates. Aptamer-NCs bioconjugates were prepared and found to be able to effectively target prostate-specific membrane antigen in a cell-specific manner.

Size-Dependent Tumor Penetration and in Vivo Efficacy of Monodisperse Drug–Silica Nanoconjugates

The size of a nanomedicine strongly correlates with its biodistribution, tissue penetration, and cell uptake. However, there is limited understanding how the size of nanomedicine impacts the overall antitumor efficacy. We designed and synthesized camptothecin-silica nanoconjugates (Cpt-NCs) with monodisperse particle sizes of 50 and 200 nm, two representative sizes commonly used in drug delivery, and evaluated their antitumor efficacy in murine tumor models. Our studies revealed that the 50 nm Cpt-NC showed higher anticancer efficacy than the larger analogue, due presumably to its faster cellular internalization and more efficient tumor accumulation and penetration. Our findings suggest that nanomedicine with smaller sizes holds great promise for improved cancer therapy.

Use of Alkaline Phosphatase Staining to Differentiate Canine Osteosarcoma from Other Vimentin-positive Tumors

Aspiration of lytic bone lesions is an excellent diagnostic test in the initial evaluation of primary bone neoplasia. However, cytologically, it can be difficult to differentiate osteosarcoma (OSA) from other bone neoplasms, including fibrosarcoma, chondrosarcoma, synovial cell sarcoma, and plasma cell myeloma. The purpose of this study is to determine the sensitivity and specificity of alkaline phosphatase (ALP) staining to differentiate OSA from other tumors that express vimentin by immunocytochemistry or immunohistochemistry. ALP is a hydrolytic enzyme present in multiple tissues including liver, kidney, intestine, placenta, and bone. Hypothetically, neoplasms actively producing bone should be specifically positive for ALP staining. Unstained, cytologic specimens were incubated for 8-10 minutes with nitroblue tetrazolium chloride/5-bromo-4-chloro-3-indolyl phosphate toluidine salt-phosphatase substrate. A positive reaction stains the membrane of the cells gray to black. Samples were counterstained with a Romanowskys stain to determine whether the sample was of representative cellularity. A total of 61 vimentin-positive neoplasms have been evaluated and confirmed histopathologically. Tumors that expressed vimentin and were positive for ALP included 33 OSAs, one multilobular tumor of bone, one amelanotic melanoma, and one chondrosarcoma. Tumors that expressed vimentin and were negative for ALP included chondrosarcomas (three of four), multiple fibrosarcomas, and multiple synovial cell sarcomas. The sensitivity is 100%, and the specificity is 89%. In conclusion, ALP appears to be a highly sensitive and fairly specific marker in the diagnosis of OSA.

Aptamer‐Functionalized, Ultra‐Small, Monodisperse Silica Nanoconjugates for Targeted Dual‐Modal Imaging of Lymph Nodes with Metastatic Tumors

Metastases are responsible for 90% of human cancer deaths.[1] Most solid tumors metastasize through the circulation system, and the sentinel lymph node (LN) is typically the first site reached by the disseminating malignant cancer cells.[2] The detection of LN metastases is therefore crucial for accurate tumor staging and therapeutic decision-making.[3] The current standard method for LN assessment is lymphography using a vital blue dye. However, this method is invasive, involving extended nodal dissection, and can give a false negative result if a LN is missed in surgery.[4] A non-invasive LN imaging technique is urgently needed to improve the accuracy of tumor staging.[5] Various techniques for sentinel LN imaging have been investigated, such as near-infrared (NIR) fluorescence imaging, computed tomography (CT), magnetic resonance imaging (MRI), positron emission tomography (PET), and ultrasound and photoacoustic imaging.[4b, 5b, 6] However, each technique has its drawbacks, and none is sufficient to provide all the necessary information for LN assessment. [4b] PET is the most sensitive and specific technique for in vivo molecular imaging,[7] but it suffers from low spatial resolution. In contrast, fluorescence imaging has high resolution and allows spatial visualization, which is helpful for intraoperative guidance; but its application is limited by poor tissue penetration. Therefore, combination of both PET and fluorescence imaging together potentially permit non-invasive assessment of LNs with high sensitivity and excellent spatial resolution.

Single-Agent Pamidronate for Palliative Therapy of Canine Appendicular Osteosarcoma Bone Pain

BACKGROUND Canine appendicular osteosarcoma (OSA) causes focal bone destruction, leading to chronic pain and reduced quality-of-life scores. Drugs that inhibit pathologic osteolysis might provide additional treatment options for managing cancer-induced bone pain. Aminobisphosphonates induce osteoclast apoptosis, thereby reducing pain associated with malignant osteolysis in human patients with cancer. HYPOTHESIS Treatment of dogs with pamidronate administered intravenously will alleviate bone pain and reduce pathologic bone turnover associated with appendicular OSA in dogs. ANIMALS Forty-three dogs with naturally occurring appendicular OSA administered pamidronate intravenously. METHODS Prospective study. Therapeutic responses in dogs treated with pamidronate administered intravenously and nonsteroidal anti-inflammatory drugs (NSAID) were evaluated by using a numerical cumulative pain index score (CPIS), and by quantifying urine N-telopeptide (NTx) excretion and relative primary tumor bone mineral density (rBMD) assessed with dual energy x-ray absorptiometry. In addition, variables, including pamidronate dose, skeletal mass, baseline and change for CPIS, urine NTx and rBMD during treatment, and baseline tumor volume and radiographic pattern were compared between dogs clinically responsive and nonresponsive to pamidronate therapy. RESULTS Twelve of 43 dogs (28%) had pain alleviation for >4 months, lasting a median of 231 days. Changes in CPIS and rBMD during treatment were statistically different between responders and nonresponders (P = .046 and .03, respectively). CONCLUSIONS AND CLINICAL IMPORTANCE Substantiated by reductions in CPIS and increases in rBMD, single-agent pamidronate administered intravenously with NSAID therapy relieves pain and diminishes pathologic bone turnover associated with appendicular OSA in a subset of dogs.

Evaluation of Intravenous Pamidronate Administration in 33 Cancer‐Bearing Dogs with Primary or Secondary Bone Involvement

The purpose of this study was to evaluate the clinical safety of pamidronate when administered at a mean dosage of 1.0 mg/kg IV q28d in 33 tumor-bearing dogs. Biochemical tests of renal function were evaluated before each successive pamidronate treatment. Of 33 dogs treated with pamidronate, 1 dog had clinically relevant increases in serum creatinine and blood urea nitrogen concentrations. The biologic activity of IV pamidronate was assessed prospectively in 10 dogs with appendicular osteosarcoma and was assessed on reductions in urine N-telopeptide excretion (P = .042) and enhanced bone mineral density of the primary tumor measured with dual-energy x-ray absorptiometry (P = .024). Additionally, in these 10 dogs, pamidronates therapeutic activity was supported by subjective improvement in pain control in 4 of the 10 dogs treated. IV pamidronate appears clinically safe in tumor-bearing dogs and may possess modest biologic activity for managing neoplastic complications associated with pathologic bone resorption.

Effect of leukoreduction on transfusion-induced inflammation in dogs

BACKGROUND Removal of leukocytes (LR) has been shown to eliminate or attenuate many of the adverse effects of transfusion in experimental animals and humans. HYPOTHESIS/OBJECTIVES Transfusion of stored packed red blood cells (pRBCs) is associated with an inflammatory response in dogs and prestorage LR attenuates the inflammatory response. ANIMALS Thirteen random-source, clinically healthy, medium and large breed dogs. METHODS Experimental study. On day 0, animals were examined and baseline blood samples were collected for analysis. Whole blood was then collected for processing with and without LR, and stored as pRBC. Twenty-one days later, stored pRBCs were transfused back to the donor. Blood samples were collected before and 1 and 3 days after transfusion. RESULTS In the dogs that received non-LR pRBCs (n = 6) there was a significant increase from baseline in white blood cell count from a mean (SD) of 8.20 (2.74) to 13.95 (4.60) × 10(3) cells/μL (P < .001) and in segmented neutrophil count from a mean (SD) of 5.76 (2.70) to 11.91 (4.71) × 10(3) cells/μL (P < .001). There were also significant increases in fibrinogen from a mean (SD) of 129.7 (24.2) to 268.6 (46.7) mg/dL (P < .001) and C-reactive protein from a mean (SD) of 1.9 (2.1) to 78.3 (39.3) μg/mL (P < .001). There was no significant increase from baseline in any of the markers in the dogs that received LR pRBC (n = 5). CONCLUSIONS AND CLINICAL IMPORTANCE There is a profound inflammatory response to transfusion in normal dogs, which is eliminated by LR of the pRBC units.

Poly(iohexol) Nanoparticles As Contrast Agents for in Vivo X-ray Computed Tomography Imaging

Biocompatible poly(iohexol) nanoparticles, prepared through cross-linking of iohexol and hexamethylene diisocyanate followed by coprecipitation of the resulting cross-linked polymer with mPEG-polylactide, were utilized as contrast agents for in vivo X-ray computed tomography (CT) imaging. Compared to conventional small-molecule contrast agents, poly(iohexol) nanoparticles exhibited substantially protracted retention within the tumor bed and a 36-fold increase in CT contrast 4 h post injection, which makes it possible to acquire CT images with improved diagnosis accuracy over a broad time frame without multiple administrations.