Neeraj Prabhakar

Design considerations for mesoporous silica nanoparticulate systems in facilitating biomedical applications

Abstract Mesoporous silica nanoparticles (MSNs) have advanced to the forefront of multifunctional nanoparticulate systems in nanomedicine, owing to this highly fexible materials platform enabling a multitude of design options, often in a modular fashion. Drug delivery ability, detectability via diferent imaging modalities, and stimuliresponsiveness are often combined into one particle system. Very sophisticated and versatile designs along with impressive demonstrations of applicability have been reported to date, but a common ground when it comes to some critical considerations valid for any nanoparticle intended for biomedical purposes is lacking to some degree. In this study, we attempt to take a glance at some of the most crucial aspects of biomedical nanoparticulate design and relate how they apply specifically toMSNs. These considerations include fuorophore labeling and leaching with respect to immobilization to MSNs, the surrounding conditions, carrier biodegradability, and surface coating. Surface modifcation strategies and surface charge tuning are further considered in conjunction to the relative amount of cellular uptake and serum protein adsorption. Cellular internalization routes and biological techniques used to evaluate especially in vitro biobehavior are discussed. Our attempt is hereby to draw attention to some of the most frequently occurring issues to be considered in the design of MSN systems for biomedical applications

Ratiometric Sensing and Imaging of Intracellular pH Using Polyethylenimine-Coated Photon Upconversion Nanoprobes

Measurement of changes of pH at various intracellular compartments has potential to solve questions concerning the processing of endocytosed material, regulation of the acidification process, and also acidification of vesicles destined for exocytosis. To monitor these events, the nanosized optical pH probes need to provide ratiometric signals in the optically transparent biological window, target to all relevant intracellular compartments, and to facilitate imaging at subcellular resolution without interference from the biological matrix. To meet these criteria we sensitize the surface conjugated pH sensitive indicator via an upconversion process utilizing an energy transfer from the nanoparticle to the indicator. Live cells were imaged with a scanning confocal microscope equipped with a low-energy 980 nm laser excitation, which facilitated high resolution and penetration depth into the specimen, and low phototoxicity needed for long-term imaging. Our upconversion nanoparticle resonance energy transfer based sensor with polyethylenimine-coating provides high colloidal stability, enhanced cellular uptake, and distribution across cellular compartments. This distribution was modulated with membrane integrity perturbing treatment that resulted into total loss of lysosomal compartments and a dramatic pH shift of endosomal compartments. These nanoprobes are well suited for detection of pH changes in in vitro models with high biological background fluorescence and in in vivo applications, e.g., for the bioimaging of small animal models.

Controlled synthesis, bioimaging and toxicity assessments in strong red emitting Mn2+ doped NaYF4:Yb3+/Ho3+ nanophosphors

Rare earth, Yb3+/Ho3+ doped NaYF4 nanophosphors showed augmentation of visible green and red light emission by the introduction of Mn2+ as a co-dopant. Nanophosphors were characterized for their morphology and upconversion (UC) fluorescence, as a function of co-dopant concentration. The effect of Mn2+ doping has been investigated to explore the UC mechanism of the phosphors. With the introduction of Mn2+, the intensity of all the emission bands was increased, in particular the red band, which was most significant at 40 mol% doping. A possible mechanism for the enhancement of the red emission has been proposed. The prepared UC nanoparticles were functionalized with polyethylene glycol–polyethylene imine co-polymer to make them water dispersible and successfully applied for cancer cell imaging.

Targeted modulation of cell differentiation in distinct regions of the gastrointestinal tract via oral administration of differently PEG-PEI functionalized mesoporous silica nanoparticles

Targeted delivery of drugs is required to efficiently treat intestinal diseases such as colon cancer and inflammation. Nanoparticles could overcome challenges in oral administration caused by drug degradation at low pH and poor permeability through mucus layers, and offer targeted delivery to diseased cells in order to avoid adverse effects. Here, we demonstrate that functionalization of mesoporous silica nanoparticles (MSNs) by polymeric surface grafts facilitates transport through the mucosal barrier and enhances cellular internalization. MSNs functionalized with poly(ethylene glycol) (PEG), poly(ethylene imine) (PEI), and the targeting ligand folic acid in different combinations are internalized by epithelial cells in vitro and in vivo after oral gavage. Functionalized MSNs loaded with γ-secretase inhibitors of the Notch pathway, a key regulator of intestinal progenitor cells, colon cancer, and inflammation, demonstrated enhanced intestinal goblet cell differentiation as compared to free drug. Drug-loaded MSNs thus remained intact in vivo, further confirmed by exposure to simulated gastric and intestinal fluids in vitro. Drug targeting and efficacy in different parts of the intestine could be tuned by MSN surface modifications, with PEI coating exhibiting higher affinity for the small intestine and PEI–PEG coating for the colon. The data highlight the potential of nanomedicines for targeted delivery to distinct regions of the tissue for strict therapeutic control.

Curcumin associated poly(allylamine hydrochloride)-phosphate self-assembled hierarchically ordered nanocapsules: size dependent investigation on release and DPPH scavenging activity of curcumin

Combination of a cationic polyamine with a multivalent anionic salt results in the spontaneous generation of ionically crosslinked capsules. Here we report curcumin associated poly(allylamine hydrochloride) crosslinks with dipotassium phosphate and subsequently congregates with silica nanoparticles to form hierarchically ordered nanocapsule structures. The capsule sizes vary depending on the concentration of dipotassium phosphate. SEM data ascertain the spherical shape of the nanocapsules and TEM analysis demonstrates that the outer layer made up of silica has a thickness of ∼50–250 nm. The fluorescence images confirm that curcumin are present all over the capsules. Strong interaction between nanocapsules and curcumin is evident from spectroscopic analysis and TGA data. Release of curcumin from the nanocapsules is found to be triggered by pH where basic environment trigger the maximum release compared to acidic and neutral conditions. The drug release profile of curcumin from the nanocapsules follows the Higuchi model and depends on the size of the nanocapsules. 2,2-Diphenyl-1-picrylhydrazyl (DPPH) scavenging activity of the encapsulated curcumin decreases exponentially with a decrease in size of the nanocapsules, suggesting not only weight percentage of curcumin in the nanocapsules plays a role but also availability of β-diketone group of curcumin for H-donation is vital. Thus, in larger nanocapsules more curcumin per surface area of the nanocapsules are exposed on the outside for scavenging activity, compared to smaller sized nanocapsules where a substantial percentage of curcumin are buried/encapsulated inside the capsules without exposing themselves for scavenging activity.

Stimuli-responsive hybrid nanocarriers developed by controllable integration of hyperbranched PEI with mesoporous silica nanoparticles for sustained intracellular siRNA delivery

Small interfering RNA (siRNA) is a highly potent drug in gene-based therapy with the challenge being to deliver it in a sustained manner. The combination of mesoporous silica nanoparticles (MSNs) and polycations in the confined pore space allows for incorporation and controlled release of therapeutic siRNA payloads. We hereby constructed MSNs with expanded mesopores and pore-surface-hyperbranched poly(ethyleneimine) (PEI) tethered with redox-cleavable linkers that could carry a high payload of siRNA (120 mg·g−1). The developed nanocarriers were efficiently taken up by cancer cells and were subsequently able to escape to the cytoplasm from the endosomes, most likely owing to the integrated PEI. Triggered by the intracellular redox conditions, the siRNA was sustainably released inside the cells over a period of several days. Functionality of siRNAs was demonstrated by using cell-killing siRNA as cargo. Despite not being the aim of the developed system, in vitro experiments using cell-killing siRNAs showed that the efficacy of siRNA transfection was comparable to the commercial in vitro transfection agent Lipofectamine. Consequently, the developed MSN-based delivery system offers a potential approach to hybrid nanocarriers for more efficient and long-term siRNA delivery and, in a longer perspective, in vivo gene silencing for RNA interference (RNAi) therapy.

Prolonged dye release from mesoporous silica-based imaging probes facilitates long-term optical tracking of cell populations in vivo

Nanomedicine is gaining ground worldwide in therapy and diagnostics. Novel nanoscopic imaging probes serve as imaging tools for studying dynamic biological processes in vitro and in vivo. To allow detectability in the physiological environment, the nanostructure-based probes need to be either inherently detectable by biomedical imaging techniques, or serve as carriers for existing imaging agents. In this study, the potential of mesoporous silica nanoparticles carrying commercially available fluorochromes as self-regenerating cell labels for long-term cellular tracking is investigated. The particle surface is organically modified for enhanced cellular uptake, the fluorescence intensity of labeled cells is followed over time both in vitro and in vivo. The particles are not exocytosed and particles which escaped cells due to cell injury or death are degraded and no labeling of nontargeted cell populations are observed. The labeling efficiency is significantly improved as compared to that of quantum dots of similar emission wavelength. Labeled human breast cancer cells are xenotransplanted in nude mice, and the fluorescent cells can be detected in vivo for a period of 1 month. Moreover, ex vivo analysis reveals fluorescently labeled metastatic colonies in lymph node and rib, highlighting the capability of the developed probes for tracking of metastasis.

On the intracellular release mechanism of hydrophobic cargo and its relation to the biodegradation behavior of mesoporous silica nanocarriers.

The intracellular release mechanism of hydrophobic molecules from surface-functionalized mesoporous silica nanoparticles was studied in relation to the biodegradation behavior of the nanocarrier, with the purpose of determining the dominant release mechanism for the studied drug delivery system. To be able to follow the real-time intracellular release, a hydrophobic fluorescent dye was used as model drug molecule. The in vitro release of the dye was investigated under varying conditions in terms of pH, polarity, protein and lipid content, presence of hydrophobic structures and ultimately, in live cancer cells. Results of investigating the drug delivery system show that the degradation and drug release mechanisms display a clear interdependency in simple aqueous solvents. In pure aqueous media, the cargo release was primarily dependent on the degradation of the nanocarrier, while in complex media, mimicking intracellular conditions, the physicochemical properties of the cargo molecule itself and its interaction with the carrier and/or surrounding media were found to be the main release-governing factors. Since the material degradation was retarded upon loading with hydrophobic guest molecules, the cargo could be efficiently delivered into live cancer cells and released intracellularly without pronounced premature release under extracellular conditions. From a rational design point of view, pinpointing the interdependency between these two processes can be of paramount importance considering future applications and fundamental understanding of the drug delivery system.

Semiconducting Polymer Encapsulated Mesoporous Silica Particles with Conjugated Europium Complexes: Toward Enhanced Luminescence under Aqueous Conditions

Immobilization of lanthanide organic complexes in meso-organized hybrid materials for luminescence applications have attracted immense interest due to the possibility of controlled segregation at the nanoscopic level for novel optical properties. Aimed at enhancing the luminescence intensity and stability of the hybrid materials in aqueous media, we developed polyvinylpyrrolidone (PVP) stabilized, semiconducting polymer (poly(9-vinylcarbazole), PVK) encapsulated mesoporous silica hybrid particles grafted with Europium(III) complexes. Monosilylated β-diketonate ligands (1-(2-naphthoyl)-3,3,3-trifluoroacetonate, NTA) were first co-condensed in the mesoporous silica particles as pendent groups for bridging and anchoring the lanthanide complexes, resulting in particles with an mean diameter of ∼ 450 nm and a bimodal pore size distribution centered at 3.5 and 5.3 nm. PVK was encapsulated on the resulted particles by a solvent-induced surface precipitation process, in order to seal the mesopores and protect Europium ions from luminescence quenching by producing a hydrophobic environment. The obtained polymer encapsulated MSN-EuLC@PVK-PVP particles exhibit significantly higher intrinsic quantum yield (Φ(Ln) = 39%) and longer lifetime (τ(obs) = 0.51 ms), as compared with those without polymer encapsulation. Most importantly, a high luminescence stability was realized when MSN-EuLC@PVK-PVP particles were dispersed in various aqueous media, showing no noticeable quenching effect. The beneficial features and positive attributes of both mesoporous silica and semiconducting polymers as lanthanide-complex host were merged in a single hybrid carrier, opening up the possibility of using these hybrid luminescent materials under complex aqueous conditions such as biological/physiological environments.

STED-TEM Correlative Microscopy Leveraging Nanodiamonds as Intracellular Dual-Contrast Markers

Development of fluorescent and electron dense markers is essential for the implementation of correlative light and electron microscopy, as dual-contrast landmarks are required to match the details in the multimodal images. Here, a novel method for correlative microscopy that utilizes fluorescent nanodiamonds (FNDs) as dual-contrast probes is reported. It is demonstrated how the FNDs can be used as dual-contrast labels-and together with automatic image registration tool SuperTomo, for precise image correlation-in high-resolution stimulated emission depletion (STED)/confocal and transmission electron microscopy (TEM) correlative microscopy experiments. It is shown how FNDs can be employed in experiments with both live and fixed cells as well as simple test samples. The fluorescence imaging can be performed either before TEM imaging or after, as the robust FNDs survive the TEM sample preparation and can be imaged with STED and other fluorescence microscopes directly on the TEM grids.