Generation of Well-Defined Micro/Nanoparticles via Advanced Manufacturing Techniques for Therapeutic Delivery

Purpose: Conventional micro/nanoparticle synthesis techniques (e.g., emulsion, sol–gel) can produce only a limited number of shapes—mainly spherical or rod-like—and provide poor control over size, cargo loading, and surface properties. These features critically affect particle–cell interactions and in vivo biodistribution, so advanced manufacturing techniques are needed to generate well-defined micro/nanoparticles for improved therapeutic delivery. ---

Hypothesis: No formal testable hypothesis is proposed. The central thesis is: if advanced manufacturing techniques impose spatial and temporal control over particle fabrication, then micro/nanoparticles with precisely defined size, shape, surface property, and component materials can be produced, and these well-controlled features can improve in vitro and in vivo performance for drug, vaccine, and cell-based therapeutic delivery. ---

Aims: - Review key micro/nanoparticle characteristics—size, surface property, component materials, shape, and toxicity—and their impact on therapeutic and imaging applications. - Review advanced manufacturing techniques for producing controllable micro/nanoparticles, including photolithography, soft lithography, nanoimprint lithography, PRINT, mechanical stretching, and microfluidic fabrication. - Discuss therapeutic delivery applications of these well-defined particles, including drug delivery, vaccine development, cell-surface engineering, and bio-imaging. - Highlight opportunities and challenges for translating advanced manufactured particles into biomedical applications. ---

Delivery system: Particle production approaches: - Bottom-up methods: conventional synthesis (emulsion, sol–gel), yielding spherical/rod shapes with limited control. - Top-down/advanced manufacturing methods: photolithography, soft lithography, nanoimprint lithography (NIL), Particle Replication In Non-wetting Templates (PRINT), mechanical stretching, microfluidic fabrication, and nanoarchitectonics. Materials used: - Synthetic polymers: PLGA, PLA, PGA, PMMA, polyelectrolytes, poly(ethylene glycol) (PEG), poly(N-isopropylacrylamide) (PNIPAM), poly(L-histidine)-PEG, PLLA/PEG-PSD. - Biological materials: polysaccharides, DNA, proteins/peptides, lipids/liposomes. - Inorganic/metallic: gold nanoparticles, iron oxide nanoparticles, mesoporous silicon, carbon nanotubes, nanodiamonds, C60 fullerenes. - Hydrogels, thermoplastic materials, photocurable perfluoropolyether (PFPE) molds. Payloads: - siRNA, DNA, mRNA, microRNA probes, antibodies, antigens, adjuvants, doxorubicin (DOX), paclitaxel (PTX), catalase, MRI contrast agents, thermal-responsive materials, fluorescence dyes, Raman reporters. Targeting/functional elements: - Antibodies (e.g., anti-DEC-205), carbohydrates, peptides, aptamers, dextran, charged surface groups (amine, carboxyl, sulfate, hydroxyl), PEGylation. Particle features engineered: - Size, shape (spherical, disk, rod, elliptical, cubic, cylindrical, needle, dot-on-pad, Janus), aspect ratio, modulus, surface charge, surface chemistry, cargo loading, and stimuli responsiveness. ---

Approach: Narrative review of published literature. No primary experimental groups. Model systems discussed include: - In vitro: Caco-2 cells, HeLa cells, macrophages, dendritic cells, T cells, antigen-presenting cells, A549 cells, human skin fibroblasts, T lymphocytes, mouse macrophages. - In vivo: Mice, tumor-bearing mice, carotid artery injection, pulmonary delivery models, inflammatory tissue models, melanoma and pancreatic cancer models. - Disease/therapeutic context: Drug delivery, vaccine development, cancer treatment, cell tracking, bio-imaging, cell-based drug delivery, inflammation. - Clinical translation: Limited; most advanced manufactured particle systems remain preclinical. ---

Key methods: Techniques highlighted across cited studies: - SEM/TEM for particle morphology and shape. - Fluorescence microscopy and confocal imaging for cellular uptake and particle–cell interactions. - Flow cytometry for internalization and immune cell analysis. - In vivo biodistribution and organ accumulation. - Cytokine secretion assays. - MRI for magnetic nanoparticle tracking. - Raman labeling for cell tracking. - Enzymatic reaction-rate measurements (Michaelis–Menten analysis for catalase-laden particles). - Cytotoxicity and cell viability assays. - Immune response assays (antigen-specific T cell activation). - Renal/hepatic biomarker and plasma cytokine analysis for biosafety. ---

Key results: - Size-dependent uptake: 100 nm particles showed 2.5-fold greater uptake by Caco-2 cells than 1 µm particles and 6-fold higher uptake than 10 µm particles. - Size-dependent internalization mechanisms: Particles >500 nm are internalized mainly by phagocytosis; <500 nm by pinocytosis. Particles <200 nm enter via clathrin-coated pits; ~500 nm via caveolae-mediated internalization. ~200 nm particles are more easily routed to lysosomes than ~500 nm particles. - In vivo size effects: >200 nm particles are more easily trapped at injection sites; <200 nm particles traffic to lymph nodes. Particles <500 nm circulate throughout the body. For capillary passage, rigid particles must be <10 µm. Airborne particles 0.5–5 µm reach deep lungs; <0.5 µm are exhaled. - Shape-dependent internalization: PRINT cylindrical particles with high aspect ratio showed almost 4-fold higher internalization rate than low-aspect-ratio particles in HeLa cells. Disk-shaped 6 µm particles resisted phagocytosis, whereas spherical particles <6 µm were quickly phagocytosed. - Advanced manufacturing resolution: NIL can achieve resolution down to 2 nm; interference lithography can produce ~50 nm features at wafer scale. - Mesoporous silicon particles: Did not change renal/hepatic biomarkers or 23 plasma cytokines; antibody-modified particles targeted pancreas/lung more effectively than unmodified particles. - Catalase-laden microdevices: Retained enzyme activity after 1 week; reaction rate followed Michaelis–Menten kinetics, enabling prediction of H₂O₂ degradation. - PRINT particles and tumor immune status: Tumor presence increased M2-like macrophages, enhanced nanoparticle clearance, and increased accumulation in liver and spleen. - Microfluidic/flow lithography: Enabled barcoded particles for multiplex miRNA assays and spatially encoded particles for drug delivery, cell tracking, and diagnostics. ---

Interpretation: The authors conclude that advanced manufacturing techniques can generate micro/nanoparticles with controllable features that enable unique biomedical applications, especially drug delivery, vaccine development, cell tracking, and bio-imaging. Particle size, shape, surface property, and component materials strongly affect cellular uptake, intracellular trafficking, in vivo circulation, biodistribution, degradation, and toxicity. Well-controlled particles offer opportunities to optimize therapeutic delivery and to study particle–immune interactions. However, deeper understanding of toxicity mechanisms and more in-depth studies are still required. ---

Limitations: - Conventional synthesis limitations: poor control over size, shape, and cargo loading. - Photolithography: requires expensive cleanroom equipment. - Nanoimprint lithography: UV exposure and reactive ion etching steps may damage therapeutic cargos such as DNA and peptides. - PRINT: advanced but requires specialized PFPE molds and fabrication expertise. - Mechanical stretching: shape control depends on polymer viscosity, interfacial tension, film thickness, and processing parameters. - Microfluidics: mostly produces spherical particles; non-spherical particle production remains limited. - In vivo complexity: biological system is complicated; particle behavior is difficult to predict. - Toxicity concerns: reactive oxygen species, oxidative stress, inflammation, autophagy/apoptosis/necrosis; toxicity mechanisms vary by cell type and are not fully understood. - Clinical translation: very limited commercially available in vivo micro/nano products. - Review limitations: not a systematic review or meta-analysis; no primary data; some citation details were not fully visible in the supplied file.