Nanochannel Delivery Systems for Controlled and Sustained Administration of Therapeutics and Cell Transplantation

Problem: Medical practitioners have long realized the need for personalized treatment that can synergize with the body’s innate response to diseases and medical conditions. Currently, the most common approaches to drug administration in medicine are 1) oral or intravenous delivery of a “bolus” (a large amount of drug delivered all at once) at regular intervals and 2) intravenous slow infusions. Bolus administration is commonly associated with adverse side effects derived from a temporary regional overdose following each administration to the patient. Infusions are required in cases of highly toxic drugs (typical of numerous chemotherapeutics), which force patients to be hospitalized for hours to days in order to monitor the severe side effects of the treatments. This conventional practice is arguably a rudimentary and impersonal approach to drug delivery, where therapeutics are often inefficiently delivered at the maximum tolerated dose (MTD). Can we instead mimic the body’s natural control over the release of molecules with accurate dosing and precise timing? The answer to personalized treatment can be found in nanotechnology and nanoscale fluid mechanics.

A conceptual design of our implantable, artificial NanoGland incorporated into a distributed biosensor network (BSN). The first inset (from left to right, top to bottom) depicts an implant prototype, containing the nanochannel membrane, the drug reservoir, sensor, telemetry, and control electronics, and a battery or RF antenna for remote powering. The second inset is an actual fabricated nanochannel membrane complete with surface deposited gold electrodes. The graph shows data (acquired at the beginning of February 2012) of the controlled release of dendritic fullerene 1 (a free radical scavenging agent) from a nanochannel membrane.

Approach: Steady-Release NanoGlands: Nanofluidic membranes reveal counterintuitive mass transport phenomena in nanochannels caused by fluid confinement. Our research in the Department of Nanomedicine has shown that by shrinking the channels by a factor close to the sizes of the diffusing molecules, it was possible to achieve a saturated, concentration-independent transport through the membranes. This shows promise for controlled transport of molecules and therapeutics. As such, we focus on the development of nanopores, nanochanneled membranes and nanoscale fluid mechanics. To advance this technology, we have used sacrificial layer techniques to reproducibly fabricate nanochannels as small as 3 nm. By exploiting nanochannels in passive systems, we were able to achieve a controlled and constant delivery of therapeutics and nanoparticles, in vitro and in vivo, for extended periods of time, while mimicking the basal and continuous release of molecules from glands to the rest of the body. This functionality cannot be attained at the macro- or microscale without the use of complex pumps and other moving components, because the diffusion of molecules is “Fickian,” which means that the release rate is dictated by the gradient of the molecular concentration.

Active-Release NanoGlands: A time-variable rate of release can also be achieved through electrokinetic phenomena that occur when an electrical potential is applied between the inlet and outlet of the channels. Electrokinetic phenomena, such as electrophoresis and electroosmosis, have been widely studied in microchannels. We have developed implantable nanochannel membranes with electrodes near the membrane inlet and outlet and demonstrated that the delivery of molecules in proportion to the applied electrical field is up to 10 times higher than for passive delivery. Additionally, by reversing the polarity of the applied electrical field, the molecule release can completely stop. As opposed to the rudimentary conventional practice where drugs are “digitally” administered, such nanochannel technologies afford “analog” time modulation, where timing, duration and frequency of administration can be precisely adjusted as needed. This approach mimics the time-variable release of molecules in the body and can enable new delivery regimens, including “chronotherapy,” the synchronization of drug delivery with optimal times during the circadian cycle of the body.

Injectable Multistage Nanovectors (MSV) for Improved Therapy and Diagnosis 

Problem: An abundance of barriers reduce the likelihood that drugs and imaging agents will reach the site of action. For drugs delivered by intravenous injection, these include enzymatic degradation, uptake by the reticuloendothelial system and crossing the endothelial barrier, cellular membranes and cellular efflux pumps. For diseases like cancer, there is an urgent need to achieve efficient concentrations of drugs in the target tissue with minimal distribution in healthy tissue. Overcoming these biological barriers, delivering one or multiple entities and personalizing therapy have historically been addressed by trying to endow individual drug molecules with one or all of these capabilities.

Approach: Nanotechnology offers unprecedented opportunities to develop treatments that increase therapeutic efficacy, decrease undesired side effects and effectively achieve the personalization of intervention for conditions, such as cancer and cardiovascular and infectious diseases. The majority of current nanotherapeutics/nanodiagnostics in clinics and under investigation accommodate single or multiple functionalities on the same entity. However, due to a multiplicity of heterogeneous biological barriers, therapeutic and imaging agents are unable to reach their intended targets in sufficient concentrations. Thus we envisioned and introduced a multistage nanovector (MSV) in which different nanocomponents (or stages) responsible for a variety of functions are decoupled but act in a synergistic manner.

Stage 1 mesoporous silicon particles (S1MP) were rationally designed and fabricated using semiconductor fabrication techniques, photolithography and electrochemical etching in a non-spherical geometry to enable superior blood margination and to increase cell surface adhesion. The main task of S1MP is to efficiently transport the payload nanoparticles, termed Stage 2 nanoparticles (S2NP), which are loaded into the porous structure. Depending on the S1MP surface modifications and porosity, a variety of S2NPs (such as liposomes, micelles, metal particles and carbon structures) or nanoparticle “cocktails” can be loaded and efficiently delivered to the disease site, enabling simultaneous functions.

The versatility of the MSV platform allows for a multiplicity of applications. For example, loading of contrast agents for magnetic resonance imaging to hemispherical and discoidal S1MP enabled a significant increase in contrast efficiency (up to 50 times compared to clinically available agents.) Furthermore, administration of a single dose of MSV loaded with nanoliposomes containing siRNA, enabled sustained gene silencing for at least 21 days and, as a result, reduced tumor burden in orthotopic ovarian cancer models. We have also shown that intracellular trafficking and cell-to-cell communication can be controlled by surface modifications of S1MP and S2NP. The therapeutic and imaging potential of MSV is being investigated in primary and disseminated tumors as well as in cardiovascular and infectious diseases. 

Schematic Summary of Possible MSV Mechanisms of Action 

Central Compartment: Hemispherical or disc-shaped nanoporous silicon S1MPs are engineered to exhibit an enhanced ability to marginate within blood vessels and adhere to disease-associated endothelium. Once positioned at the disease site, the S1MP can (top right) release the drug/siRNA-loaded S2NP to achieve the desired therapeutic effect, prior to the complete biodegradation of the carrier particle; release an imaging agent (top left) or external energy-activated S2NP (e.g., gold nanoparticles, nanoshells, bottom right). Another possible mechanism of action is cell-based delivery of the MSVs into the disease loci followed by triggered release of the S1MP/S2NP from the cells.

The field of proteomics is being actively investigated to tap the clinical potential of proteins and peptides that can be used as biological markers. The most challenging technical hurdle obstructing the discovery of new protein biomarker candidates is the ability to acquire access to the most clinically relevant circulating proteomes in the blood. Our group has developed nanoporous silica chips that utilize nanoscale pores to capture high molecular-weight proteins/peptides from complex entities such as serum and plasma.

BioNanoScaffolds for Bone Tissue Engineering 

Problem: The current clinical “gold standard” for repair of critical size bone fractures is the autologous bone graft, which has shown some success but leaves the patient with secondary complications at the site of the donor tissue harvest. Other treatments include allografts (tissue taken from cadavers) and plastic or metal implants. Allografts are often biologically rejected and carry a serious risk of immune rejection or disease transmission. Synthetic implants are usually permanent, do not promote tissue regeneration and are mechanically incompatible with native tissue. This compliance mismatch leads to mechanical failure or erosion of the neighboring bone. New endeavors with tissue engineering use biodegradable scaffolding materials to direct tissue regeneration. However, this has shown limited success due to the multiple facets of tissue regeneration. Generally, polymers may differ in strength, compression and torsion, and they may not promote cell growth and tissue infiltration. Soft materials that are biocompatible may lack the mechanical properties necessary for orthopedic applications. To resolve these principle issues, we devised a strategy using bionanoscaffolds (BNS) that combines the mechanical advantages of biodegradable synthetic polymers with the biological functions of natural biomaterial scaffolds. This approach achieves the correct strength requirements while enhancing the regeneration of healthy bone tissue at the fracture site.

Approach: Bionanoscaffolds for post-traumatic osteoregeneration are a new class of composites, biologically active fracture putty materials, consisting of several fundamental building blocks. The principle components of the BNS include the following:

• Biodegradable, biocompatible polymeric scaffolds fabricated as nonporous elements for the mechanical stabilization of segmental defects
• A bioactive biomimetic osteogenic sponge that integrates factor-releasing particles to promote immediate angiogenesis and rapid regeneration of bone tissue within the defect; this scaffold can be enriched with multi-substituted hydroxyapatite crystals, which enhance bone deposition and accelerate healing 
• Nanoporous silicon enclosures formulated to control the release of bioactive molecules and factors able to accelerate healing and promote tissue reconstruction while fighting infection and biofilm formation 
• Self-assembling amphiphilic peptides that stimulate the regeneration of bone and soft tissue, including neural and vascular functions 
• Mesenchymal stem cells are adult stem cells harvested from cortical bone, marrow or adipose tissue that are capable of differentiating into mature skeletal cells and regenerating orthopedic tissue matrix
  
  

The BioNanoScaffold approach for bone tissue engineering. (A) A scaffold based on synthetic and natural polymers is implanted or injected into the fracture site. (B) The scaffold is made of highly porous, biomimetic collagen organized in randomly distributed fibers, enriched with hydroxyapatite crystals and alginate microspheres, and embedded with stem cells and delivery platforms for growth factors, differentiating stimuli, and antibiotics. (C) Upon implantation, the alginate spheres start degrading, progressively releasing their contents within the scaffold. The synergy between stem cells and growth and differentiating factors induces the reorganization of the scaffold. Osteoblasts remodel the collagen fibers and lay down additional extracellular matrix, and osteoid and hydroxyapatite crystals. (D) After a month, the fracture is healed and the implanted materials have been resorbed.

The BNS can be formulated into an injectable paste for irregular defects or space-maintaining filler in cranio-facial, spine and other fracture repairs. This material is based on calcium alginate microbeads that embed and protect all of the components as previously mentioned during delivery to the patient. Upon injection, the alginate beads progressively degrade over time, releasing a framework of biological factors, cellular components, and bioactive compounds that promote osteogenesis and early vascularization. The alginate beads can be tailored by size, porosity and overall stability, so that their degradation time and mechanical properties can be adjusted to those required by the specific application.

Problem 1: Although currently available biotechnology has provided us with a greater understanding of the molecular events in cancer, integrated microfluidic chips provide unique methodologies that can recapitulate the spatial and temporal control of cell proliferation and cell-cell/matrix communication by combining surfaces that mimic the complex biochemistries and geometries of the extracellular matrix.

Approach 1: Microfluidics research under Lidong Qin, PhD, focuses on developing integrated proteomic microchips to analyze cell heterogeneity using state-of-the-art bioinformatics tools and identifying metastatic signatures. The research aims are to deliver brand-new technologies and methodologies capable of identifying tumor-initiating cells, discovering potential biomarkers for clinical diagnosis and targeted therapy, and identifying cancer patients with metastatic propensity.

Typical Microfluidics Device. (A) An autocad drawing to show our design of single cell barcode chip (SCBC). This schematic picture presents 100 horizontal channels x 36 vertical valves and creates 3600 microchambers for the single-cell experiment. Chamber number could expand to 300 x 50 = 15,000 for a PDMS device bonding to a 75 mm x 50 mm glass substrate. (B) A typical scanned protein barcode image zoomed from a portion of the SCBC device. The protein expression level from single cell or a few cell chambers is measured by fluorescent intensity shown as red bars in between green bar pairs. Yellow numbers represent cell number counted from the experiment, resulting in the barcode image.

Problem 2: Inspired by a global need for better diagnosis and treatment strategies, this research focuses on point-of-care (POC) challenges to ultimately design a flexible multiplatform microfluidic device for the rapid and direct characterization of rare, disease-related cells for individualized patient care.

Approach 2: We intend to revolutionize disease diagnosis and treatment strategies globally through the following measures: 

• Fabricating selective and sensitive microfluidic devices that can detect antigens secreted by rare, disease-related cells on a single cell level at concentrations below the sub-picomolar level
• Correlating amounts of secreted antigens with aggression/metastatic behaviors of the disease by on-chip quantitative analysis using fluorescence for more personalized care 
• Facilitating 10 or more different drug treatments simultaneously on a single microfluidic chip in 24 hours 
• Scaling up production, delivering devices worldwide and commencing preclinical studies at the community level in resource-poor settings through overseas collaborations for fast delivery of cutting edge technology to those who need it most