Justin Hanes

Mucus-penetrating nanoparticles for drug and gene delivery to mucosal tissues☆

Mucus is a viscoelastic and adhesive gel that protects the lung airways, gastrointestinal (GI) tract, vagina, eye and other mucosal surfaces. Most foreign particulates, including conventional particle-based drug delivery systems, are efficiently trapped in human mucus layers by steric obstruction and/or adhesion. Trapped particles are typically removed from the mucosal tissue within seconds to a few hours depending on anatomical location, thereby strongly limiting the duration of sustained drug delivery locally. A number of debilitating diseases could be treated more effectively and with fewer side effects if drugs and genes could be more efficiently delivered to the underlying mucosal tissues in a controlled manner. This review first describes the tenacious mucus barrier properties that have precluded the efficient penetration of therapeutic particles. It then reviews the design and development of new mucus-penetrating particles that may avoid rapid mucus clearance mechanisms, and thereby provide targeted or sustained drug delivery for localized therapies in mucosal tissues.

Rapid transport of large polymeric nanoparticles in fresh undiluted human mucus

Nanoparticles larger than the reported mesh-pore size range (10–200 nm) in mucus have been thought to be much too large to undergo rapid diffusional transport through mucus barriers. However, large nanoparticles are preferred for higher drug encapsulation efficiency and the ability to provide sustained delivery of a wider array of drugs. We used high-speed multiple-particle tracking to quantify transport rates of individual polymeric particles of various sizes and surface chemistries in samples of fresh human cervicovaginal mucus. Both the mucin concentration and viscoelastic properties of these cervicovaginal samples are similar to those in many other human mucus secretions. Unexpectedly, we found that large nanoparticles, 500 and 200 nm in diameter, if coated with polyethylene glycol, diffused through mucus with an effective diffusion coefficient (Deff) only 4- and 6-fold lower than that for the same particles in water (at time scale τ = 1 s). In contrast, for smaller but otherwise identical 100-nm coated particles, Deff was 200-fold lower in mucus than in water. For uncoated particles 100–500 nm in diameter, Deff was 2,400- to 40,000-fold lower in mucus than in water. Much larger fractions of the 100-nm particles were immobilized or otherwise hindered by mucus than the large 200- to 500-nm particles. Thus, in contrast to the prevailing belief, these results demonstrate that large nanoparticles, if properly coated, can rapidly penetrate physiological human mucus, and they offer the prospect that large nanoparticles can be used for mucosal drug delivery.

Oral drug delivery with polymeric nanoparticles: The gastrointestinal mucus barriers

Oral delivery is the most common method for drug administration. However, poor solubility, stability, and bioavailability of many drugs make achieving therapeutic levels via the gastrointestinal (GI) tract challenging. Drug delivery must overcome numerous hurdles, including the acidic gastric environment and the continuous secretion of mucus that protects the GI tract. Nanoparticle drug carriers that can shield drugs from degradation and deliver them to intended sites within the GI tract may enable more efficient and sustained drug delivery. However, the rapid secretion and shedding of GI tract mucus can significantly limit the effectiveness of nanoparticle drug delivery systems. Many types of nanoparticles are efficiently trapped in and rapidly removed by mucus, making controlled release in the GI tract difficult. This review addresses the protective barrier properties of mucus secretions, how mucus affects the fate of orally administered nanoparticles, and recent developments in nanoparticles engineered to penetrate the mucus barrier.

Micro- and macrorheology of mucus.

Mucus is a complex biological material that lubricates and protects the human lungs, gastrointestinal (GI) tract, vagina, eyes, and other moist mucosal surfaces. Mucus serves as a physical barrier against foreign particles, including toxins, pathogens, and environmental ultrafine particles, while allowing rapid passage of selected gases, ions, nutrients, and many proteins. Its selective barrier properties are precisely regulated at the biochemical level across vastly different length scales. At the macroscale, mucus behaves as a non-Newtonian gel, distinguished from classical solids and liquids by its response to shear rate and shear stress, while, at the nanoscale, it behaves as a low viscosity fluid. Advances in the rheological characterization of mucus from the macroscopic to nanoscopic levels have contributed critical understanding to mucus physiology, disease pathology, and the development of drug delivery systems designed for use at mucosal surfaces. This article reviews the biochemistry that governs mucus rheology, the macro- and microrheology of human and laboratory animal mucus, rheological techniques applied to mucus, and the importance of an improved understanding of the physical properties of mucus to advancing the field of drug and gene delivery.

Addressing the PEG Mucoadhesivity Paradox to Engineer Nanoparticles that “Slip” through the Human Mucus Barrier

Mucus linings serve as the bodys first line of defense at exposed surfaces of the eye and respiratory, gastrointestinal, and cervicovaginal tracts. The high viscoelasticity and adhesivity of mucus traps and limits exposure to foreign pathogens,[1,2] toxins,[3] and environmental ultrafine particles,[1,4] which are all typically removed by normal mucus clearance mechanisms. Numerous studies have demonstrated that human mucus also strongly immobilizes conventional synthetic nanoparticles,[5-7] and therefore represents a hurdle for localized drug and gene delivery at mucosal surfaces, such as aerosol-based gene carriers for cystic fibrosis gene therapy.[8] To increase the bioavailability of cargo therapeutics, it is important that carrier particles rapidly penetrate mucus to avoid being shed.

Efficient active transport of gene nanocarriers to the cell nucleus

The intracellular transport of therapeutic gene carriers is poorly understood, limiting the rational design of efficient new vectors. We used live-cell real-time multiple particle tracking to quantify the intracellular transport of hundreds of individual nonviral DNA nanocarriers with 5-nm and 33-ms resolution. Unexpected parallels between several of natures most efficient DNA viruses and nonviral polyethylenimine/DNA nanocomplexes were revealed to include motor protein-driven transport through the cytoplasm toward the nucleus on microtubules. Active gene carrier transport led to efficient perinuclear accumulation within minutes. The results provide direct evidence to dispute the common belief that the efficiency of nonviral gene carriers is dramatically reduced because of the need for their relatively slow random diffusion through the cell cytoplasm to the nucleus and, instead, focuses the attention of rational carrier design on overcoming barriers downstream of perinuclear accumulation.

Biodegradable polymer nanoparticles that rapidly penetrate the human mucus barrier

Protective mucus coatings typically trap and rapidly remove foreign particles from the eyes, gastrointestinal tract, airways, nasopharynx, and female reproductive tract, thereby strongly limiting opportunities for controlled drug delivery at mucosal surfaces. No synthetic drug delivery system composed of biodegradable polymers has been shown to penetrate highly viscoelastic human mucus, such as non-ovulatory cervicovaginal mucus, at a significant rate. We prepared nanoparticles composed of a biodegradable diblock copolymer of poly(sebacic acid) and poly(ethylene glycol) (PSA-PEG), both of which are routinely used in humans. In fresh undiluted human cervicovaginal mucus (CVM), which has a bulk viscosity approximately 1,800-fold higher than water at low shear, PSA-PEG nanoparticles diffused at an average speed only 12-fold lower than the same particles in pure water. In contrast, similarly sized biodegradable nanoparticles composed of PSA or poly(lactic-co-glycolic acid) (PLGA) diffused at least 3,300-fold slower in CVM than in water. PSA-PEG particles also rapidly penetrated sputum expectorated from the lungs of patients with cystic fibrosis, a disease characterized by hyperviscoelastic mucus secretions. Rapid nanoparticle transport in mucus is made possible by the efficient partitioning of PEG to the particle surface during formulation. Biodegradable polymeric nanoparticles capable of overcoming human mucus barriers and providing sustained drug release open significant opportunities for improved drug and gene delivery at mucosal surfaces.

Nanoparticles reveal that human cervicovaginal mucus is riddled with pores larger than viruses

The mechanisms by which mucus helps prevent viruses from infecting mucosal surfaces are not well understood. We engineered non-mucoadhesive nanoparticles of various sizes and used them as probes to determine the spacing between mucin fibers (pore sizes) in fresh undiluted human cervicovaginal mucus (CVM) obtained from volunteers with healthy vaginal microflora. We found that most pores in CVM have diameters significantly larger than human viruses (average pore size 340 ± 70 nm; range approximately 50–1800 nm). This mesh structure is substantially more open than the 15–100-nm spacing expected assuming mucus consists primarily of a random array of individual mucin fibers. Addition of a nonionic detergent to CVM caused the average pore size to decrease to 130 ± 50 nm. This suggests hydrophobic interactions between lipid-coated “naked” protein regions on mucins normally cause mucin fibers to self-condense and/or bundle with other fibers, creating mucin “cables” at least three times thicker than individual mucin fibers. Although the native mesh structure is not tight enough to trap most viruses, we found that herpes simplex virus (approximately 180 nm) was strongly trapped in CVM, moving at least 8,000-fold slower than non-mucoadhesive 200-nm nanoparticles. This work provides an accurate measurement of the pore structure of fresh, hydrated ex vivo CVM and demonstrates that mucoadhesion, rather than steric obstruction, may be a critical protective mechanism against a major sexually transmitted virus and perhaps other viruses.

A Dense Poly(Ethylene Glycol) Coating Improves Penetration of Large Polymeric Nanoparticles Within Brain Tissue

Nanoparticles densely coated with poly(ethylene glycol) rapidly penetrate within mouse, rat, and human brain parenchyma. Brain-Penetrating Particles It was once thought that particles larger than 60 nm would be stuck in the brain extracellular space (ECS), unable to penetrate further. This has been a particularly bothersome rule of thumb for the design of drug delivery systems that rely on larger particles or viruses to carry therapeutics. Now, Nance and colleagues have challenged this hypothesis by exploring particles that are >60 nm, discovering that large particles, with the right coating, can indeed diffuse throughout the ECS of both rat and human brains. The authors first coated fluorescent polystyrene particles with a dense layer of the bio-inert polymer poly(ethylene glycol) (commonly known as PEG) or with a carboxyl moiety (COOH). Using a multiple-particle tracking method, the authors reported that 114-nm PEG-coated particles penetrated ex vivo human brain tissue with ease, whereas similarly sized COOH-coated particles were stopped in their tracks. Nance et al. attributed this difference to the dense, near-neutral PEG coating, claiming that it allows the particles to experience the brain ECS more as a fluid than an impermeable solid. The importance of the PEG coating was further confirmed in living mice, where real-time video microscopy revealed that the 100-nm PEG-coated particles penetrated farther into the mouse brain than the 100-nm COOH-coated ones. With a brain ECS pore size cutoff >100 nm, many doors can be opened in nanomedicine. Larger particles permit the inclusion of higher quantities of drug, which can be distributed for longer periods of time to more areas within the brain. Nance and colleagues preliminarily demonstrated such drug delivery capabilities using paclitaxel-loaded, 85-nm biodegradable nanoparticles, showing that only particles with the PEG coating could diffuse rapidly throughout rat brain tissue ex vivo. Although these densely coated particles may make drug delivery more efficient, they have yet to be tested in a disease model to confirm efficacy over conventional nanoparticles. Although currently limited to direct infusion into the brain, for eventual use in humans, it is hoped that they may be administered systemically for treating diseases with an impaired blood-brain barrier. Prevailing opinion suggests that only substances up to 64 nm in diameter can move at appreciable rates through the brain extracellular space (ECS). This size range is large enough to allow diffusion of signaling molecules, nutrients, and metabolic waste products, but too small to allow efficient penetration of most particulate drug delivery systems and viruses carrying therapeutic genes, thereby limiting effectiveness of many potential therapies. We analyzed the movements of nanoparticles of various diameters and surface coatings within fresh human and rat brain tissue ex vivo and mouse brain in vivo. Nanoparticles as large as 114 nm in diameter diffused within the human and rat brain, but only if they were densely coated with poly(ethylene glycol) (PEG). Using these minimally adhesive PEG-coated particles, we estimated that human brain tissue ECS has some pores larger than 200 nm and that more than one-quarter of all pores are ≥100 nm. These findings were confirmed in vivo in mice, where 40- and 100-nm, but not 200-nm, nanoparticles spread rapidly within brain tissue, only if densely coated with PEG. Similar results were observed in rat brain tissue with paclitaxel-loaded biodegradable nanoparticles of similar size (85 nm) and surface properties. The ability to achieve brain penetration with larger nanoparticles is expected to allow more uniform, longer-lasting, and effective delivery of drugs within the brain, and may find use in the treatment of brain tumors, stroke, neuroinflammation, and other brain diseases where the blood-brain barrier is compromised or where local delivery strategies are feasible.

Enhanced viscoelasticity of human cystic fibrotic sputum correlates with increasing microheterogeneity in particle transport.

Current biochemical characterizations of cystic fibrosis (CF) sputum do not address the high degree of microheterogeneity in the rheological properties of the mucosal matrix and only provide bulk-average particle diffusion coefficients. The viscoelasticity of CF sputum greatly reduces the diffusion rates of colloidal particles, limiting the effectiveness of gene delivery to underlying lung cells. We determine diffusion coefficients of hundreds of individual amine-modified and carboxylated polystyrene particles (diameter 100–500 nm) embedded in human CF sputum with 5 nm and 33 ms of spatiotemporal resolution. High resolution multiple particle tracking is used to calculate the effective viscoelastic properties of CF sputum at the micron scale, which we relate to its macroscopic viscoelasticity. CF sputum microviscosity, as probed by 100- and 200-nm particles, is an order of magnitude lower than its macroviscosity, suggesting that nanoparticles dispersed in CF sputum are transported primarily through lower viscosity pores within a highly elastic matrix. Multiple particle tracking provides a non-destructive, highly sensitive method to quantify the high heterogeneity of the mucus pore network. The mean diffusion coefficient becomes dominated by relatively few but fast-moving particles as particle size is reduced from 500 to 100 nm. Neutrally charged particles with a diameter <200 nm undergo more rapid transport in CF sputum than charged particles. Treatment with recombinant human DNase (Pulmozyme®) reduces macroviscoelastic properties of CF sputum by up to 50% and dramatically narrows the distribution of individual particle diffusion rates but surprisingly does not significantly alter the ensemble-average particle diffusion rate.