Degradable Microparticles Research Articles

Sustained release of inactivated H1N1 virus from degradable microparticles for extended vaccination

Influenza vaccines administered as intramuscularly injected inactivated viruses or intranasally administered live-attenuated viruses usually provide short-term protection against influenza infections. Biodegradable particles that provide sustained release of the antigen has been studied as an approach to extend vaccine protection. Here, we investigate sustained release of ultraviolet killed influenza A virus (A/PR/8/34(H1N1)) (kPR8) loaded into poly(D,L-lactic-co-glycolic acid) (PLGA) microparticles. Particles were prepared using the double emulsion method, and polymer molecular weight (MW), polymer hydrophobicity, polymer concentration in the organic phase, and the amount of killed virus were varied to obtain a range of particles. Formulations included PLGA 50:50 (2–6, 7–17 kDa), PLGA 75:25 (4–15 kDa), and 50/50 PLGA 75:25 (4–15 kDa)/PCL (14 kDa). Additionally, NaOH was co-encapsulated in some cases to enhance particle degradation. The structure of the particles was explored by size measurements and electron microscopy. The kPR8 release profiles were measured using hemagglutinin ELISA. The concentration of the polymer (PLGA) in the organic phase and polymer MW significantly influenced virus loading, while polymer MW and co-encapsulation of NaOH modulated the release profiles. Mice receiving a single intramuscular injection of NaOH microparticle-encapsulated kPR8 were partially protected against a lethal influenza challenge 32 weeks post immunization. Microparticle (MP) vaccination induced a gradual increase in PR8-specific IgGs dominated by IgG1 in contrast to the rapid IgG2a-biased response elicited by soluble kPR8 immunization. Our results indicate that vaccine-NaOH co-loaded PLGA particles show potential as a single dose vaccination strategy for extended protection against influenza virus infection.

Sustained release system from PLGA particles co-encapsulated with inactivated influenza virus with natural killer T cell agonist α-galactosylceramide

Vaccines against influenza and many other infectious diseases require multiple boosters in addition to the primary dose to improve efficacy, but this approach is not ideal for compliance. The multiple doses could potentially be replaced by sustained or pulsatile release of antigens encapsulated in degradable microparticles (MPs). The efficacy of a vaccine is improved by adding an adjuvant, which can be co-delivered from the particles to enhance immunogenicity. Here, we developed degradable poly-lactic-co-glycolic acid (PLGA) (7–17 kDa) MPs capable of sustained release of ultraviolet killed influenza virus (A/PR/8/34) (kPR8) vaccine and the natural killer T (NKT) cell agonist alpha-galactosylceramide (α-GalCer) and tested their effectiveness at providing long-term protection against influenza virus infection in mice. Multiple formulations were developed for encapsulating the virus and adjuvant separately, and in combination. The MPs exhibited sustained release of both the virus and the adjuvant lasting more than a month. Co-encapsulation significantly increased the encapsulation efficiency (EE) of the vaccine but reduced the release duration. On the other hand, co-encapsulation led to a reduction in EE for the α-GalCer and a change in release profile to a higher initial burst followed by a linear release compared to a low initial burst and slower linear release. The α-GalCer also had considerably longer release duration compared to the vaccine. Mice injected with particle formulations co-encapsulating kPR8 and α-GalCer were protected from a lethal influenza virus infection 30 weeks after vaccination. This study demonstrates that PLGA MP based vaccines are promising for providing effective vaccination and possibly for replacing multiple doses with a single injection.

Engineering the lymph node environment promotes antigen-specific efficacy in type 1 diabetes and islet transplantation

Antigen-specific tolerance is a key goal of experimental immunotherapies for autoimmune disease and allograft rejection. This outcome could selectively inhibit detrimental inflammatory immune responses without compromising functional protective immunity. A major challenge facing antigen-specific immunotherapies is ineffective control over immune signal targeting and integration, limiting efficacy and causing systemic non-specific suppression. Here we use intra-lymph node injection of diffusion-limited degradable microparticles that encapsulate self-antigens with the immunomodulatory small molecule, rapamycin. We show this strategy potently inhibits disease during pre-clinical type 1 diabetes and allogenic islet transplantation. Antigen and rapamycin are required for maximal efficacy, and tolerance is accompanied by expansion of antigen-specific regulatory T cells in treated and untreated lymph nodes. The antigen-specific tolerance in type 1 diabetes is systemic but avoids non-specific immune suppression. Further, microparticle treatment results in the development of tolerogenic structural microdomains in lymph nodes. Finally, these local structural and functional changes in lymph nodes promote memory markers among antigen-specific regulatory T cells, and tolerance that is durable. This work supports intra-lymph node injection of tolerogenic microparticles as a powerful platform to promote antigen-dependent efficacy in type 1 diabetes and allogenic islet transplantation.

Kirigami-inspired stents for sustained local delivery of therapeutics.

Implantable drug depots have the capacity to locally meet therapeutic requirements by maximizing local drug efficacy and minimizing potential systemic side effects. Tubular organs including the gastrointestinal tract, respiratory tract and vasculature all manifest with endoluminal disease. The anatomic distribution of localized drug delivery for these organs using existing therapeutic modalities is limited. Application of local depots in a circumferential and extended longitudinal fashion could transform our capacity to offer effective treatment across a range of conditions. Here we report the development and application of a kirigami-based stent platform to achieve this. The stents comprise a stretchable snake-skin-inspired kirigami shell integrated with a fluidically driven linear soft actuator. They have the capacity to deposit drug depots circumferentially and longitudinally in the tubular mucosa of the gastrointestinal tract across millimetre to multi-centimetre length scales, as well as in the vasculature and large airways. We characterize the mechanics of kirigami stents for injection, and their capacity to engage tissue in a controlled manner and deposit degradable microparticles loaded with therapeutics by evaluating these systems ex vivo and in vivo in swine. We anticipate such systems could be applied for a range of endoluminal diseases by simplifying dosing regimens while maximizing drug on-target effects through the sustained release of therapeutics and minimizing systemic side effects.

A polyphenolic biomacromolecule prepared from a flavonoid: Catechin as degradable microparticles

AbstractCatechin (CAT) was crosslinked with trimethylolpropane triglycidyl ether (TMPTGE) to obtain degradable poly(CAT) particles in a single step. Spherical p(CAT) particles with tens of micrometer size range and an isoelectronic point at pH 1.2 were obtained. The hydrolytic degradation of p(CAT) particles provided sustainable and extended release with 264 mg/g CAT release within 10 days at pH 7.4. The antioxidant capacity of 55.0 ± 0.9 μg/ml gallic acid equivalent in terms of total phenol content, and 0.88 ± 0.3 μmol/g trolox equivalent were estimated for p(CAT) particles displaying strong radical scavenging capability. Blood clotting and hemolysis assays demonstrated dose‐dependent blood compatibility revealing higher blood compatibility for p(CAT) particles up to 10 μg/ml concentration. The cytotoxicity results show that p(CAT) particles have almost no toxicity for CCD841 normal colon cells at 250 μg/ml concentration in 24 h incubation time giving ~97% cell viability, whereas CAT molecules only provide ~34% cell viability.

The bright and the dark side of the sphere: light-stabilized microparticles

We introduce degradable microparticles, synthesized from prepolymers in a precipitation-like polymerization. The narrow disperse particles are stabilized with continuous irradiation of green light and can be spontaneously degraded in the dark.

Abstract 3922: Biodistribution of Ra-224 and its daughter Pb-212 after intraperitoneal infusion of Ra-224 labeled microparticles in rats

Abstract Introduction: Radium-224 (Ra-224) labeled calcium carbonate (CaCO3) microparticles were developed with the intent to treat micrometastases located in the abdominal cavity. The slowly degradable microparticles act as carriers for the α-emitter Ra-224 and ensure high intraperitoneal (IP) retention of the radioactivity without cellular targeting. This novel α-therapy has a short action range in tissue and is designed to confine the radiation exposure to the IP cavity, treating both linings of the peritoneal surfaces and liquid volumes. The distribution of the Ra-224 labeled microparticles was examined by planar gamma imaging, SPECT and CT. The ex vivo biodistribution of Ra-224 and its progeny Pb-212 was also determined. Experimental procedures: Fifteen female Wistar rats were infused IP with Ra-224-labeled microparticles (200-400 kBq, 100 mg CaCO3). The injections were performed by use of a perforated catheter (pigtail catheter), followed by a Plasmalyte flush to mimic the planned clinical administration. The Ra-224 labeled microparticles were imaged using both planar gamma imaging, SPECT and CT to evaluate the distribution over time in the abdominal region. Ex vivo biodistribution was performed 2, 24, 96, 168 and 336 hours after particle infusion and organs were harvested for activity measurements. The activity was determined at 2 hours and 48 hours after harvesting for all samples. This allowed differentiation between the biodistribution of Ra-224 and the biodistribution of its progeny Pb-212. Furthermore, 4 rats were injected IP with free Ra-224 and 2 rats were injected intravenously with free Ra-224 to achieve a baseline for the biodistribution. These control animals were euthanized at 24 and 168 hours, and 168 hours, respectively. Results: Based on both CT and SPECT images, the particles distributed to the entire peritoneum, albeit with local areas of high microparticle concentration. Over the course of the experiment there was, as expected, a modest but evident systemic leakage of Ra-224 from the particles in the peritoneal cavity. The amount was estimated by the bone uptake of radioisotopes Pb-212 and Ra-224, using IP administered free Ra-224 as reference. The retention of Ra-224 in the peritoneal cavity was found to be > 87% at 24 hours and > 77% at 168 hours. Redistribution of the progeny Pb-212 was observed as modest uptake in the kidneys. Conclusions: High peritoneal retention of both Ra-224 and its progeny Pb-212 after IP injection of Ra-224 labeled CaCO3 microparticles, was found in rats with high translational value to the clinical setting. SPECT imaging supported distribution of the Ra-224 labeled microparticles to the entire peritoneal lumen in the animals. SPECT and CT revealed some clusters of labeled microparticles. Due to the short range of the therapeutic relevant α-particles, the clusters are not expected to have an impact on therapeutic or safety aspects. Citation Format: Jesper Fonslet, Carsten H. Nielsen, Lotte K. Kristensen, Ida S. Jorstad, Kim Lindland, Roy H. Larsen, Øyvind S. Bruland, Andreas Kjaer, Tina B. Bønsdorff. Biodistribution of Ra-224 and its daughter Pb-212 after intraperitoneal infusion of Ra-224 labeled microparticles in rats [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3922.

Targeted protein delivery: carbodiimide crosslinking influences protein release from microparticles incorporated within collagen scaffolds.

Tissue engineering response may be tailored via controlled, sustained release of active agents from protein-loaded degradable microparticles incorporated directly within three-dimensional (3D) ice-templated collagen scaffolds. However, the effects of covalent crosslinking during scaffold preparation on the availability and release of protein from the incorporated microparticles have not been explored. Here, we load 3D ice-templated collagen scaffolds with controlled additions of poly-(DL-lactide-co-glycolide) microparticles. We probe the effects of subsequent N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride crosslinking on protein release, using microparticles with different internal protein distributions. Fluorescein isothiocyanate labelled bovine serum albumin is used as a model protein drug. The scaffolds display a homogeneous microparticle distribution, and a reduction in pore size and percolation diameter with increased microparticle addition, although these values did not fall below those reported as necessary for cell invasion. The protein distribution within the microparticles, near the surface or more deeply located within the microparticles, was important in determining the release profile and effect of crosslinking, as the surface was affected by the carbodiimide crosslinking reaction applied to the scaffold. Crosslinking of microparticles with a high proportion of protein at the surface caused both a reduction and delay in protein release. Protein located within the bulk of the microparticles, was protected from the crosslinking reaction and no delay in the overall release profile was seen.

Sustained antimicrobial activity and reduced toxicity of oxidative biocides through biodegradable microparticles.

The manuscript explores a novel approach to utilize the antimicrobial activity of oxidative species for sustained killing of multiple drug-resistant organisms without causing collateral tissue damage. The results demonstrate, for the first time, the ability to load pre-cursor compounds into porous polymeric structures that results in their release and conversion into oxidative species in a controlled manner. Until now, the use of oxidative species has not been considered as a candidate therapeutic replacement for conventional antibiotics due to difficulties associated with handling during manufacture and controlling sustained release without causing undesirable tissue damage. The ultimate impact of the research could be the creation of new materials-based anti-infective chemotherapeutic agents that have minimal potential for giving rise to antimicrobial resistance.

Shape‐Memory Capability of Copolyetheresterurethane Microparticles Prepared via Electrospraying

Multifunctional thermo‐responsive and degradable microparticles exhibiting a shape‐memory effect (SME) have attracted widespread interest in biomedicine as switchable delivery vehicles or microactuators. In this work almost spherical solid microparticles with an average diameter of 3.9 ± 0.9 μm are prepared via electrospraying of a copolyetheresterurethane named PDC, which is composed of crystallizable oligo(p‐dioxanone) (OPDO) hard and oligo(ε‐caprolactone) (OCL) switching segments. The PDC microparticles are programmed via compression at different pressures and their shape‐memory capability is explored by off‐line and online heating experiments. When a low programming pressure of 0.2 MPa is applied a pronounced thermally‐induced shape‐memory effect is achieved with a shape recovery ratio about 80%, while a high programming pressure of 100 MPa resulted in a weak shape‐memory performance. Finally, it is demonstrated that an array of PDC microparticles deposited on a polypropylene (PP) substrate can be successfully programmed into a smart temporary film, which disintegrates upon heating to 60 °C.

In silico programming of degradable microparticles to hide and then reveal immunogenic payloads in vivo.

The ability to deliver but hide immunogenic payloads and then reveal them at predetermined times could lead to autonomously boosting vaccine formulations or improved antigen-adjuvant vaccine designs. We used in silico modeling to determine the appropriate formulation and material properties for poly(lactic-co-glycolic) acid (PLGA) microparticles such that they would delay the in vitro"unmasking" of an ovalbumin-alum payload for precise and predetermined intervals. A preferred formulation was then tested in vivo. In vivo T cell proliferation data confirmed the presentation of antigen released through the programmed delayed burst while antibody subclass data demonstrated immunogenicity comparable to that observed with established multiple injection prime-boost regimens.

Correlating the Uptake and Dendritic Cell Activation by MDP-loaded Microparticles

ABSTRACTPolymer-based, degradable microparticles (MP) are attractive delivery vehicles for vaccines as the polymer properties can be specifically tailored and the carrier can be loaded with adjuvant. For all newly developed carrier systems it is important to analyze cellular uptake efficiency and the specific effects mediated by the encapsulated agent when phagocytosed by the cells, which is barely reported so far. By the encapsulation of N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP) labeled with fluoresceinisothiocyanat (FITC) in poly[(rac-lactide)-co-glycolide] (PLGA) MP, the MP was fluorescent and used to visualize the phagocytic uptake. Since encapsulated MDP can activate dendritic cells (DC) via the cytosolic nucleotide-binding oligomerization domain receptors (NOD), it can be investigated whether only cells that have phagocytosed the MP are activated or whether bystander effects occur, resulting in activation of cells, which did not take up MDP-FITC loaded MP. Here, it is demonstrated that increasing MP concentrations in the culture medium had no impact on the viability of DC and that the MP uptake efficiency was dose dependent. Interestingly, it could be shown by the CD86 expression, that only DC, which had engulfed MP, were significantly stronger activated than DC, which had not phagocytosed MDP-FITC loaded MP. On the one hand these results indicate that sufficient amounts of MDP were released from the PLGA carriers into the cytosol of the DC. On the other hand, based on the correlation of uptake and activation on the single cell level, minimal MP induced bystander effects may be expected for in vivo applications.

Enzymatic triggered release of an HIV-1 entry inhibitor from prostate specific antigen degradable microparticles

This paper describes the design, construction and characterization of the first anti-HIV drug delivery system that is triggered to release its contents in the presence of human semen. Microgel particles were synthesized with a crosslinker containing a peptide substrate for the seminal serine protease prostate specific antigen (PSA) and were loaded with the HIV-1 entry inhibitor sodium poly(styrene-4-sulfonate) (pSS). The particles were composed of N-2-hydroxyproplymethacrylamide and bis-methacrylamide functionalized peptides based on the PSA substrates GISSFYSSK and GISSQYSSK. Exposure to human seminal plasma (HSP) degraded the microgel network and triggered the release of the entrapped antiviral polymer. Particles with the crosslinker composed of the substrate GISSFYSSK showed 17 times faster degradation in seminal plasma than that of the crosslinker composed of GISSQYSSK. The microgel particles containing 1 mol% GISSFYSSK peptide crosslinker showed complete degradation in 30 h in the presence of HSP at 37 °C and pSS released from the microgels within 30 min reached a concentration of 10 μg/mL, equivalent to the published IC 90 for pSS. The released pSS inactivated HIV-1 in the presence of HSP. The solid phase synthesis of the crosslinkers, preparation of the particles by inverse microemulsion polymerization, HSP-triggered release of pSS and inactivation of HIV-1 studies are described.

Preparation of Monodisperse Biodegradable Polymer Microparticles Using a Microfluidic Flow‐Focusing Device for Controlled Drug Delivery

Degradable microparticles have broad utility as vehicles for drug delivery and form the basis of several therapies approved by the US Food and Drug Administration. Conventional emulsion-based methods of manufacturing produce particles with a wide range of diameters (and thus kinetics of release) in each batch. This paper describes the fabrication of monodisperse, drug-loaded microparticles from biodegradable polymers using the microfluidic flow-focusing (FF) devices and the drug-delivery properties of those particles. Particles are engineered with defined sizes, ranging from 10 microm to 50 microm. These particles are nearly monodisperse (polydispersity index = 3.9%). A model amphiphilic drug (bupivacaine) is incorporated within the biodegradable matrix of the particles. Kinetic analysis shows that the release of the drug from these monodisperse particles is slower than that from conventional methods of the same average size but a broader distribution of sizes and, most importantly, exhibit a significantly lower initial burst than that observed with conventional particles. The difference in the initial kinetics of drug release is attributed to the uniform distribution of the drug inside the particles generated using the microfluidic methods. These results demonstrate the utility of microfluidic FF for the generation of homogenous systems of particles for the delivery of drugs.

Delivery of rapamycin to dendritic cells using degradable microparticles

Degradable microparticles have the potential to protect and release drugs over extended periods and, if sized appropriately, can be passively targeted to phagocytic cells in vivo. Dendritic cells (DC) are a class of phagocytic cells known to play important roles in transplant rejection. Previously, we have demonstrated that DC treated with an immunosuppressive drug, rapamycin, have the ability to suppress transplant rejection. Herein, we describe a strategy to deliver an intracellular depot of rapamycin to DC. To achieve this, rapamycin was encapsulated into ~ 3.4 μm sized poly(lactic-co-glycolic)acid (PLGA) microparticles (rapaMPs), and release behavior was examined under intra-phagosomal (pH = 5) and extracellular (pH = 7.4) conditions. It was observed that 4 days following phagocytosis of rapaMP, DC have significantly reduced ability to activate T cells, in comparison to DC treated with soluble rapamycin. Hence, we conclude that DC-specific intracellular delivery of rapamycin results in better efficacy of the drug, with respect to its ability to modulate DC function, when compared to treating DC with extracellular rapamycin.

Delivery of rapamycin to dendritic cells using degradable microparticles

Degradable microparticles have the potential to protect and release drugs over extended periods and, if sized appropriately, can be targeted passively to phagocytic cells in vivo. Dendritic cells (DCs) are a class of phagocytic cells that are known to play important roles in transplant rejection. Previously, we have demonstrated that DCs treated with an immunosuppressive drug, rapamycin, have the ability to suppress transplant rejection. We have devised a strategy to deliver an intracellular depot of rapamycin to DCs. To achieve this, rapamycin was encapsulated into ∼3.4 μm sized Poly‐lactic‐co‐glycolic acid (PLGA) microparticles (rapaMPs), and release behavior examined under intra‐phagosomal (pH = 5) and extracellular (pH = 7.4) conditions. We observe that rapaMPs are internalized by DCs in large numbers, and that they do not induce DC death. Further, we demonstrate that the DCs that take up rapaMPs have significantly reduced ability to activate T cells, in comparison to DCs treated with soluble rapamycin. Hence, we conclude that DC specific intracellular delivery of rapamycin results in better efficacy of the drug (with respect to the ability to modulate DC function) when compared to treating DCs with extracellular rapamycin. We are currently in the process of investigating the hypothesis that rapaMP‐loaded DCs can be used to suppress transplant rejection.

Potent Antigen-specific Immune Responses Stimulated by Codelivery of CpG ODN and Antigens in Degradable Microparticles

CpG ODN stimulates a TH1 response through its receptor Toll-like receptor 9 (TLR9). TLR9 is a receptor that is found intracellularly. Microparticles are efficiently internalized by dendritic cells (DCs) and macrophages and would thus be an ideal delivery vehicle for CpG ODN to reach its target site thereby enhancing the TH1 response to an antigen also encapsulated in the microparticle. Here, we show that careful control over fabrication parameters can produce biodegradable microparticles with predictable size distributions, surface morphology, and shape. Entrapment efficiencies of the model antigen OVA ranged from 19% to 23% with an average loading of 10 microg/mg of microparticles. For CpG ODN, these values were 33% to 35%, which corresponded to an average loading of 8.5 microg/mg of microparticles. The microparticles release CpG ODN and OVA in a burst followed by sustained release profile. At the highest concentration of microparticles incubated with a pure DC cell line, 92% of DCs had internalized microparticles by 16 hours, confirming that DCs efficiently take up the microparticles. Microparticles are capable of inducing DC maturation as determined by up-regulation of CD80 and CD86 markers. Although the presence of CpG ODN in the microparticles did not impact on the phenotype of the DCs, it was necessary for DCs to induce activation of antigen-specific T cells as indicated by interferon-gamma production. Microparticles entrapping both antigen and CpG ODN induced significantly higher amounts of anti-OVA antibody production than other preparations such as the soluble OVA and CpG ODN (P<0.01) and stimulated stronger IgG2a production than delivery of microparticles entrapping antigen alone. We conclude that co-encapsulating immunostimulatory CpG ODN and antigen in degradable microparticles is an effective approach to enhancing development of a TH1 immune response.

Novel hepatitis B vaccines

Currently available hepatitis B vaccines are immunogenic, efficacious and safe. There is no doubt that their consistent use makes the elimination of hepatitis B in most countries possible. Nevertheless, there are still aspects of these vaccines which could be improved: three doses are needed for a full course of vaccination (which is sometimes difficult to achieve because of poor compliance or difficult logistic situations in some regions), there is a comparably high rate of non-responders to the vaccine (about 5% in adults) and, finally, it is not impossible that there are strains of HBV showing mutations of HBsAg which could escape the immunity induced by present vaccines. Work is underway to overcome these problems. Combined vaccines such as those providing protection against diphtheria, pertussis, tetanus, Haemophiulus influenza type b, hepatitis B and poliomyelitis are an important step in simplifying hepatitis B vaccination of infants. Combined vaccines are already in use of under clinical evaluation. In an effort to reduce the number of injections, two dose schedules with vaccines of higher dosages are being examined and single-dose vaccines encapsulated in degradable micro-particles of biopolymers (called slow-release vaccines) have been successfully tested in animals. Intense research is being conducted to enhance the immunogenicity of present vaccines. One possibility under investigation is the development of recombinant vaccines containing the complete preS1 and preS2 regions of HBsAg or immunogenic epitopes of these regions in addition to the small surface protein; several of such vaccines have already been tested in clinical trials. Future developments include expression of HBsAg determinants in bacteria, e.g. Salmonella, for oral vaccination or DNA vaccines against hepatitis B.

Inflammatory response to polyacryl starch microparticles, role of arachidonic acid metabolites

The effect of polyacryl starch microparticles on arachidonic acid metabolism in macrophage cultures and in mice was studied. When macrophages were incubated with the microparticles, significant amounts of arachidonic acid metabolites were released. Two of the major metabolites were identified and found to be prostaglandin E 2 and leukotriene C 4. Intravenous administration in mice of slowly degradable polyacryl starch microparticles, having a mean diameter of 1 μm, resulted in hepatomegaly and occasionally, inflammatory granulomas in the liver. Rapidly degradable microparticles did not give the same response. When the mice were treated with inhibitors of arachidonic acid metabolism, i.e. indomethacin or timegadine, the microparticle-induced hepatomegaly was partly inhibited. The microparticle-mediated effects were compared with those obtained with a well-characterized macrophageactivating/inflammatory agent, 1,3-β-glucan particles.