Immunostimulatory TLR7 agonist-nanoparticles together with checkpoint blockade for effective cancer immunotherapy.

Mono- or dual-checkpoint inhibitors for immunotherapy have changed the paradigm of cancer care; however, only a minority of patients responds to such treatment. Combining small molecule immuno-stimulators can improve treatment efficacy, but they are restricted by poor pharmacokinetics. In this study, TLR7 agonists conjugated onto silica nanoparticles showed extended drug localization after intratumoral injection. The nanoparticle-based TLR7 agonist increased immune stimulation by activating the TLR7 signaling pathway. When treating CT26 colon cancer, nanoparticle conjugated TLR7 agonists increased T cell infiltration into the tumors by > 4× and upregulated expression of the interferon γ gene compared to its unconjugated counterpart by ~2×. Toxicity assays established that the conjugated TLR7 agonist is a safe agent at the effective dose. When combined with checkpoint inhibitors that target programmed cell death protein 1 (PD-1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), a 10-100× increase in immune cell migration was observed; furthermore, 100 mm3 tumors were treated and a 60% remission rate was observed including remission at contralateral non-injected tumors. The data show that nanoparticle based TLR7 agonists are safe and can potentiate the effectiveness of checkpoint inhibitors in immunotherapy resistant tumor models and promote a long-term specific memory immune function.

Conjugation of a Small-Molecule TLR7 Agonist to Silica Nanoshells Enhances Adjuvant Activity.

Stimulation of Toll-like receptors (TLRs) and/or NOD-like receptors on immune cells initiates and directs immune responses that are essential for vaccine adjuvants. The small-molecule TLR7 agonist, imiquimod, has been approved by the FDA as an immune response modifier but is limited to topical application due to its poor pharmacokinetics that causes undesired adverse effects. Nanoparticles are increasingly used with innate immune stimulators to mitigate side effects and enhance adjuvant efficacy. In this study, a potent small-molecule TLR7 agonist, 2-methoxyethoxy-8-oxo-9-(4-carboxybenzyl)adenine (1V209), was conjugated to hollow silica nanoshells (NS). Proinflammatory cytokine (IL-6, IL-12) release by mouse bone-marrow-derived dendritic cells and human peripheral blood mononuclear cells revealed that the potency of silica nanoshells-TLR7 conjugates (NS-TLR) depends on nanoshell size and ligand coating density. Silica nanoshells of 100 nm diameter coated with a minimum of ∼6000 1V209 ligands/particle displayed 3-fold higher potency with no observed cytotoxicity when compared to an unconjugated TLR7 agonist. NS-TLR activated the TLR7-signaling pathway, triggered caspase activity, and stimulated IL-1ß release, while neither unconjugated TLR7 ligands nor silica shells alone produced IL-1ß. An in vivo murine immunization study, using the model antigen ovalbumin, demonstrated that NS-TLR increased antigen-specific IgG antibody induction by 1000× with a Th1-biased immune response, compared to unconjugated TLR7 agonists. The results show that the TLR7 ligand conjugated to silica nanoshells is capable of activating an inflammasome pathway to enhance both innate immune-stimulatory and adjuvant potencies of the TLR7 agonist, thereby broadening applications of innate immune stimulators.

Identifying lost surgical needles with visible and near infrared fluorescent light emitting microscale coating.

BACKGROUND: Retained foreign bodies (RFOs) have substantial clinical and financial consequences. In laparoscopic surgery, RFOs can be a cause of needing to convert a minimally invasive surgery (MIS) procedure to an open operation. A coating for surgical models was developed to augment localization of needles using fluorescence appropriate for open and minimally invasive surgeries procedures. METHODS: An epoxy matrix containing both dansyl chloride and indocyanine green was coated as visible and near infrared labels, respectively. With ultraviolet excitation, dansyl chloride emits green fluorescence and with NIR excitation, the ICG dye emits radiation observable with specialized near infrared capable laparoscopes. To evaluate the coatings, open and laproscopic surgeries were simulated in rabbits. Surgeons blinded to the type of needles (coated or non-coated) were timed while finding needles in standard conditions and with the use of the adjunct coatings. Control needles not located within 300 seconds were researched with the corresponding near infrared or ultraviolet light. Localization time was evaluated for statistical significance, P < .05. RESULTS: All dual dye coated needles searched utilizing the near infrared camera (n = 26) or ultraviolet light (n= 26) were located within 300 seconds. Conversely, 9 needles in both control settings (no dye usage) were not located within 300 seconds. Mean time to locate control needles in open surgery and laparoscopic surgery was statistically 2-3× greater than time to localization with the use of dye as an adjunct (P = .0027 open, P < .001 laparoscopic). CONCLUSION: Incorporation of a dual-dye fluorescent coating on surgical needles improved the efficiency of locating needles, may minimize the need to convert minimally invasive surgeries procedures to open, and may decrease the consequences of a missed RFO.

Silica Shells/Adhesive Composite Film for Color Doppler Ultrasound Guided Needle Placement.

Ultrasound (US) guided medical devices placement is a widely used clinical technology, yet many factors affect the visualization of these devices in the human body. In this research, an ultrasound-activated film was developed that can be coated on the surface of medical devices. The film contains 2 µm silica microshells and poly(methyl 2-cyanoacrylate) (PMCA) adhesive. The air sealed in the hollow space of the microshells acted as the US contrast agent. Ozone and perfluorooctyltriethoxysilane (PFO) were used to treat the surface of the film to enhance the US signals and provide durable antifouling properties for multiple passes through tissue, consistent with the dual oleophobic and hydrophobic nature of PFO. In vitro and in vivo tests showed that hypodermic needles and tumor marking wires coated with US activated film gave strong and persistent color Doppler signals. This technology can significantly improve the visibility of medical devices and the accuracy of US guided medical device placement.

Ultrasound Responsive Macrophase-Segregated Microcomposite Films for in Vivo Biosensing.

Ultrasound imaging is a safe, low-cost, and in situ method for detecting in vivo medical devices. A poly(methyl-2-cyanoacrylate) film containing 2 µm boron-doped, calcined, porous silica microshells was developed as an ultrasound imaging marker for multiple medical devices. A macrophase separation drove the gas-filled porous silica microshells to the top surface of the polymer film by controlled curing of the cyanoacrylate glue and the amount of microshell loading. A thin film of polymer blocked the wall pores of the microshells to seal air in their hollow core, which served as an ultrasound contrast agent. The ultrasound activity disappeared when curing conditions were modified to prevent the macrophase segregation. Phase segregated films were attached to multiple surgical tools and needles and gave strong color Doppler signals in vitro and in vivo with the use of a clinical ultrasound imaging instrument. Postprocessing of the simultaneous color Doppler and B-mode images can be used for autonomous identification of implanted surgical items by correlating the two images. The thin films were also hydrophobic, thereby extending the lifetime of ultrasound signals to hours of imaging in tissues by preventing liquid penetration. This technology can be used as a coating to guide the placement of implantable medical devices or used to image and help remove retained surgical items.

Assessment of in vivo systemic toxicity and biodistribution of iron-doped silica nanoshells.

Silica nanoparticles are an emerging class of biomaterials which may be used as diagnostic and therapeutic tools for biomedical applications. In particular, hollow silica nanoshells are attractive due to their hollow core. Approximately 70% of a 500 nm nanoshell is hollow, therefore more particles can be administered on a mg/kg basis compared to solid nanoparticles. Additionally, their nanoporous shell permits influx/efflux of gases and small molecules. Since the size, shape, and composition of a nanoparticle can dramatically alter its toxicity and biodistribution, the toxicology of these nanomaterials was assessed. A single dose toxicity study was performed in vivo to assess the toxicity of 500 nm iron-doped silica nanoshells at clinically relevant doses of 10-20 mg/kg. This study showed that only a trace amount of silica was detected in the body 10 weeks post-administration. The hematology, biochemistry and pathological results show that the nanoshells exhibit no acute or chronic toxicity in mice.

Utilization of iron (III)-doped nanoshells for in vivo marking of nonpalpable tumors using a VX2 rabbit model.

BACKGROUND: We aimed to evaluate the potential for ultrasound (US) visible biodegradable nanoshells (NS) as an alternative to wire-guided localization for nonpalpable tumors in vivo. METHODS: VX2 tumor was injected in bilateral thighs of 22 New Zealand rabbits and after 5 to 10 days, 1 tumor was marked with a wire as a control and the contralateral tumor was injected with 1 mL of 500 nm gas-filled silica NS under Doppler US. Tumors were excised after 24 hours. Chi-square was used for significance, P = .05. RESULTS: One rabbit was excluded on postoperative day 1 due to equipment failure, no ill effects were observed from the NS. The NS were used to localize and resect 100% of marked tissue, 4/21 wires were displaced (P < .05). CONCLUSIONS: We have shown that preoperatively injected US visible silica NS can be successfully used to mark nonpalpable tumors in vivo more consistently than WL.

Quantification of endocytosis using a folate functionalized silica hollow nanoshell platform.

A quantification method to measure endocytosis was designed to assess cellular uptake and specificity of a targeting nanoparticle platform. A simple N -hydroxysuccinimide ester conjugation technique to functionalize 100-nm hollow silica nanoshell particles with fluorescent reporter fluorescein isothiocyanate and folate or polyethylene glycol (PEG) was developed. Functionalized nanoshells were characterized using scanning electron microscopy and transmission electron microscopy and the maximum amount of folate functionalized on nanoshell surfaces was quantified with UV-Vis spectroscopy. The extent of endocytosis by HeLa cervical cancer cells and human foreskin fibroblast (HFF-1) cells was investigated in vitro using fluorescence and confocal microscopy. A simple fluorescence ratio analysis was developed to quantify endocytosis versus surface adhesion. Nanoshells functionalized with folate showed enhanced endocytosis by cancer cells when compared to PEG functionalized nanoshells. Fluorescence ratio analyses showed that 95% of folate functionalized silica nanoshells which adhered to cancer cells were endocytosed, while only 27% of PEG functionalized nanoshells adhered to the cell surface and underwent endocytosis when functionalized with 200 and 900 µg , respectively. Additionally, the endocytosis of folate functionalized nanoshells proved to be cancer cell selective while sparing normal cells. The developed fluorescence ratio analysis is a simple and rapid verification/validation method to quantify cellular uptake between datasets by using an internal control for normalization.

Synthesis and surface functionalization of silica nanoparticles for nanomedicine.

There are a wide variety of silica nanoformulations being investigated for biomedical applications. Silica nanoparticles can be produced using a wide variety of synthetic techniques with precise control over their physical and chemical characteristics. Inorganic nanoformulations are often criticized or neglected for their poor tolerance; however, extensive studies into silica nanoparticle biodistributions and toxicology have shown that silica nanoparticles may be well tolerated, and in some case are excreted or are biodegradable. Robust synthetic techniques have allowed silica nanoparticles to be developed for applications such as biomedical imaging contrast agents, ablative therapy sensitizers, and drug delivery vehicles. This review explores the synthetic techniques used to create and modify an assortment of silica nanoformulations, as well as several of the diagnostic and therapeutic applications.

Encapsulation of adenovirus serotype 5 in anionic lecithin liposomes using a bead-based immunoprecipitation technique enhances transfection efficiency.

Oncolytic viruses (OVs) constitute a promising class of cancer therapeutics which exploit validated genetic pathways known to be deregulated in many cancers. To overcome an immune response and to enhance its potential use to treat primary and metastatic tumors, a method for liposomal encapsulation of adenovirus has been developed. The encapsulation of adenovirus in non-toxic anionic lecithin-cholesterol-PEG liposomes ranging from 140 to 180 nm in diameter have been prepared by self-assembly around the viral capsid. The encapsulated viruses retain their ability to infect cancer cells. Furthermore, an immunoprecipitation (IP) technique has shown to be a fast and effective method to extract non-encapsulated viruses and homogenize the liposomes remaining in solution. 78% of adenovirus plaque forming units were encapsulated and retained infectivity after IP processing. Additionally, encapsulated viruses have shown enhanced transfection efficiency up to 4 × higher compared to non-encapsulated Ads. Extracting non-encapsulated viruses from solution may prevent an adverse in vivo immune response and may enhance treatment for multiple administrations.