Mechanisms and Barriers in Nanomedicine: Progress in the Field and Future Directions.

In recent years, steady progress has been made in synthesizing and characterizing engineered nanoparticles, resulting in several approved drugs and multiple promising candidates in clinical trials. Regulatory agencies such as the Food and Drug Administration and the European Medicines Agency released important guidance documents facilitating nanoparticle-based drug product development, particularly in the context of liposomes and lipid-based carriers. Even with the progress achieved, it is clear that many barriers must still be overcome to accelerate translation into the clinic. At the recent conference workshop "Mechanisms and Barriers in Nanomedicine" in May 2023 in Colorado, U.S.A., leading experts discussed the formulation, physiological, immunological, regulatory, clinical, and educational barriers. This position paper invites open, unrestricted, nonproprietary discussion among senior faculty, young investigators, and students to trigger ideas and concepts to move the field forward.

Tackling myelin deficits in neurodevelopmental disorders using drug delivery systems.

Interest in myelin and its roles in almost all brain functions has been greatly increasing in recent years, leading to countless new studies on myelination, as a dominant process in the development of cognitive functions. Here, we explore the unique role myelin plays in the central nervous system and specifically discuss the results of altered myelination in neurodevelopmental disorders. We present parallel developmental trajectories involving myelination that correlate with the onset of cognitive impairment in neurodevelopmental disorders and discuss the key challenges in the treatment of these chronic disorders. Recent developments in drug repurposing and nano/micro particle-based therapies are reviewed as a possible pathway to circumvent some of the main hurdles associated with early intervention, including patient's adherence and compliance, side effects, relapse, and faster route to possible treatment of these disorders. The strategy of drug encapsulation overcomes drug solubility and metabolism, with the possibility of drug targeting to a specific compartment, reducing side effects upon systemic administration.

REVIEW: "ISCHEMIC STROKE: From Fibrinolysis to Functional Recovery" Nanomedicine: Emerging Approaches to Treat Ischemic Stroke.

Stroke is responsible for 11% of all deaths worldwide, the majority of which are caused by ischemic strokes, thus making the need to urgently find safe and effective therapies. Today, these can be cured either by mechanical thrombectomy when the thrombus is accessible, or by intravenous injection of fibrinolytics. However, the latter present several limitations, such as potential severe side effects, few eligible patients and low rate of partial and full recovery. To design safer and more effective treatments, nanomedicine appeared in this medical field a few decades ago. This review will explain why nanoparticle-based therapies and imaging techniques are relevant for ischemic stroke management. Then, it will present the different nanoparticle types that have been recently developed to treat this pathology. It will also study the various targeting strategies used to bring nanoparticles to the stroke site, thereby limiting side effects and improving the therapeutic efficacy. Finally, this review will present the few clinical studies testing nanomedicine on stroke and discuss potential causes for their scarcity.

Compartmentalized drug localization studies in extracellular vesicles for anticancer therapy.

In the development of therapeutic extracellular vesicles (EVs), drug encapsulation efficiencies are significantly lower when compared with synthetic nanomedicines. This is due to the hierarchical structure of the EV membrane and the physicochemical properties of the candidate drug (molecular weight, hydrophilicity, lipophilicity, and so on). As a proof of concept, here we demonstrated the importance of drug compartmentalization in EVs as an additional parameter affecting the therapeutic potential of drug-loaded EVs. In human adipose mesenchymal stem cell (hADSC) derived EVs, we performed a comparative drug loading analysis using two formulations of the same chemotherapeutic molecule - free doxorubicin (DOX) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) lipid-conjugated doxorubicin (L-DOX) - to enhance the intracellular uptake and therapeutic efficacy. By nano surface energy transfer (NSET) and molecular simulation techniques, along with cryo-TEM analysis, we confirmed the differential compartmentalization of these two molecules in hADSC EVs. L-DOX was preferentially adsorbed onto the surface of the EV, due to its higher lipophilicity, whereas free DOX was mostly encapsulated within the EV core. Also, the L-DOX loaded EV (LDOX@EV) returned an almost three-fold higher DOX content as compared to the free DOX loaded EV (DOX@EV), for a given input mass of drug. Based on the cellular investigations, L-DOX@EV showed higher cell internalization than DOX@EV. Also, in comparison with free L-DOX, the magnitude of therapeutic potential enhancement displayed by the surface compartmentalized L-DOX@EV is highly promising and can be exploited to overcome the sensitivity of many potential drugs, which are impermeable in nature. Overall, this study illustrates the significance of drug compartmentalization in EVs and how this could affect intracellular delivery, loading efficiency, and therapeutic effect. This will further lay the foundation for the future systematic investigation of EV-based biotherapeutic delivery platforms for personalized medicine.

Nanomedicines to treat rare neurological disorders: The case of Krabbe disease.

The brain remains one of the most challenging therapeutic targets due to the low and selective permeability of the blood-brain barrier and complex architecture of the brain tissue. Nanomedicines, despite their relatively large size compared to small molecules and nucleic acids, are being heavily investigated as vehicles to delivery therapeutics into the brain. Here we elaborate on how nanomedicines may be used to treat rare neurodevelopmental disorders, using Krabbe disease (globoid cell leukodystrophy) to frame the discussion. As a monogenetic disorder and lysosomal storage disease affecting the nervous system, the lessons learned from examining nanoparticle delivery to the brain in the context of Krabbe disease can have a broader impact on the treatment of various other neurodevelopmental and neurodegenerative disorders. In this review, we introduce the epidemiology and genetic basis of Krabbe disease, discuss current in vitro and in vivo models of the disease, as well as current therapeutic approaches either approved or at different stage of clinical developments. We then elaborate on challenges in particle delivery to the brain, with a specific emphasis on methods to transport nanomedicines across the blood-brain barrier. We highlight nanoparticles for delivering therapeutics for the treatment of lysosomal storage diseases, classified by the therapeutic payload, including gene therapy, enzyme replacement therapy, and small molecule delivery. Finally, we provide some useful hints on the design of nanomedicines for the treatment of rare neurological disorders.

Matrix metalloproteinase responsive hydrogel microplates for programmed killing of invasive tumour cells.

Interactive materials are an emerging class of systems that can offer control over response and adaptivity in polymer structures towards the meso- and macroscale. Here, we use enzyme regulated cleavage of peptide crosslinkers in polymer hydrogels to release a cytotoxic therapeutic nanoparticle with an adaptable mechanism. Hydrogel microplates were formed through polyethylene glycol/peptide photoinitiated thiol-ene chemistry in a soft-lithography process to give square plates of 20 by 20 µm with a height of 10 µm. The peptide was chosen to be degradable in the presence of matrix metalloproteinase 2/9 (MMP-2/9). The hydrogel material's mechanical properties, swelling, and protease degradation were characterised. The microfabricated hydrogels were loaded with docetaxel (DTXL) containing poly(dl-lactide-co-glycolide) (PLGA) nanoparticles, and characterised for enzyme responsivity, and toxicity to MMP-2/9 overexpressing brain cancer cell line U87-MG. A 5-fold decrease in EC50 was seen compared to free DTXL, and a 20-fold decrease was seen for the MMP responsive microplates versus a non-degradable control microplate. Potential applications of this system in post-resection glioblastoma treatment are envisioned.

Machine learning instructed microfluidic synthesis of curcumin-loaded liposomes.

The association of machine learning (ML) tools with the synthesis of nanoparticles has the potential to streamline the development of more efficient and effective nanomedicines. The continuous-flow synthesis of nanoparticles via microfluidics represents an ideal playground for ML tools, where multiple engineering parameters - flow rates and mixing configurations, type and concentrations of the reagents - contribute in a non-trivial fashion to determine the resultant morphological and pharmacological attributes of nanomedicines. Here we present the application of ML models towards the microfluidic-based synthesis of liposomes loaded with a model hydrophobic therapeutic agent, curcumin. After generating over 200 different liposome configurations by systematically modulating flow rates, lipid concentrations, organic:water mixing volume ratios, support-vector machine models and feed-forward artificial neural networks were trained to predict, respectively, the liposome dispersity/stability and size. This work presents an initial step towards the application and cultivation of ML models to instruct the microfluidic formulation of nanoparticles.

Microstructured Polymeric Fabrics Modulating the Paracrine Activity of Adipose-Derived Stem Cells.

The deposition of stem cells at sites of injury is a clinically relevant approach to facilitate tissue repair and angiogenesis. However, insufficient cell engraftment and survival require the engineering of novel scaffolds. Here, a regular network of microscopic poly(lactic-co-glycolic acid) (PLGA) filaments was investigated as a promising biodegradable scaffold for human Adipose-Derived Stem Cell (hADSC) tissue integration. Via soft lithography, three different microstructured fabrics were realized where 5 × 5 and 5 × 3 µm PLGA 'warp' and 'weft' filaments crossed perpendicularly with pitch distances of 5, 10 and 20 µm. After hADSC seeding, cell viability, actin cytoskeleton, spatial organization and the secretome were characterized and compared to conventional substrates, including collagen layers. On the PLGA fabric, hADSC re-assembled to form spheroidal-like structures, preserving cell viability and favoring a nonlinear actin organization. Moreover, the secretion of specific factors involved in angiogenesis, the remodeling of the extracellular matrix and stem cell homing was favored on the PLGA fabric as compared to that which occurred on conventional substrates. The paracrine activity of hADSC was microstructure-dependent, with 5 µm PLGA fabric enhancing the expression of factors involved in all three processes. Although more studies are needed, the proposed PLGA fabric would represent a promising alternative to conventional collagen substrates for stem cell implantation and angiogenesis induction.

µMESH-Enabled Sustained Delivery of Molecular and Nanoformulated Drugs for Glioblastoma Treatment.

Modest tissue penetrance, nonuniform distribution, and suboptimal release of drugs limit the potential of intracranial therapies against glioblastoma. Here, a conformable polymeric implant, µMESH, is realized by intercalating a micronetwork of 3 × 5 µm poly(lactic-co-glycolic acid) (PLGA) edges over arrays of 20 × 20 µm polyvinyl alcohol (PVA) pillars for the sustained delivery of potent chemotherapeutic molecules, docetaxel (DTXL) and paclitaxel (PTXL). Four different µMESH configurations were engineered by encapsulating DTXL or PTXL within the PLGA micronetwork and nanoformulated DTXL (nanoDTXL) or PTXL (nanoPTXL) within the PVA microlayer. All four µMESH configurations provided sustained drug release for at least 150 days. However, while a burst release of up to 80% of nanoPTXL/nanoDTXL was documented within the first 4 days, molecular DTXL and PTXL were released more slowly from µMESH. Upon incubation with U87-MG cell spheroids, DTXL-µMESH was associated with the lowest lethal drug dose, followed by nanoDTXL-µMESH, PTXL-µMESH, and nanoPTXL-µMESH. In orthotopic models of glioblastoma, µMESH was peritumorally deposited at 15 days post-cell inoculation and tumor proliferation was monitored via bioluminescence imaging. The overall animal survival increased from ∼30 days of the untreated controls to 75 days for nanoPTXL-µMESH and 90 days for PTXL-µMESH. For the DTXL groups, the overall survival could not be defined as 80% and 60% of the animals treated with DTXL-µMESH and nanoDTXL-µMESH were still alive at 90 days, respectively. These results suggest that the sustained delivery of potent drugs properly encapsulated in conformable polymeric implants could halt the proliferation of aggressive brain tumors.

Assessing Differential Particle Deformability under Microfluidic Flow Conditions.

Assessing the mechanical behavior of nano- and micron-scale particles with complex shapes is fundamental in drug delivery. Although different techniques are available to quantify the bulk stiffness in static conditions, there is still uncertainty in assessing particle deformability in dynamic conditions. Here, a microfluidic chip is designed, engineered, and validated as a platform to assess the mechanical behavior of fluid-borne particles. Specifically, potassium hydroxide (KOH) wet etching was used to realize a channel incorporating a series of micropillars (filtering modules) with different geometries and openings, acting as microfilters in the direction of the flow. These filtering modules were designed with progressively decreasing openings, ranging in size from about 5 down to 1 µm. Discoidal polymeric nanoconstructs (DPNs), with a diameter of 5.5 µm and a height of 400 nm, were realized with different poly(lactic-co-glycolic acid) (PLGA) and poly(ethylene glycol) (PEG) ratios (PLGA/PEG), namely, 5:1 and 1:0, resulting in soft and rigid particles, respectively. Given the peculiar geometry of DPNs, the channel height was kept to 5 µm to limit particle tumbling or flipping along the flow. After thorough physicochemical and morphological characterization, DPNs were tested within the microfluidic chip to investigate their behavior under flow. As expected, most rigid DPNs were trapped in the first series of pillars, whereas soft DPNs were observed to cross multiple filtering modules and reach the micropillars with the smallest opening (1 µm). This experimental evidence was also supported by computational tools, where DPNs were modeled as a network of springs and beads immersed in a Newtonian fluid using the smoothed particle hydrodynamics (SPH) method. This preliminary study presents a combined experimental-computational framework to quantify, compare, and analyze the characteristics of particles having complex geometrical and mechanical attributes under flow conditions.

Nonspherical ultrasound microbubbles.

Surface tension provides microbubbles (MB) with a perfect spherical shape. Here, we demonstrate that MB can be engineered to be nonspherical, endowing them with unique features for biomedical applications. Anisotropic MB were generated via one-dimensionally stretching spherical poly(butyl cyanoacrylate) MB above their glass transition temperature. Compared to their spherical counterparts, nonspherical polymeric MB displayed superior performance in multiple ways, including i) increased margination behavior in blood vessel-like flow chambers, ii) reduced macrophage uptake in vitro, iii) prolonged circulation time in vivo, and iv) enhanced blood-brain barrier (BBB) permeation in vivo upon combination with transcranial focused ultrasound (FUS). Our studies identify shape as a design parameter in the MB landscape, and they provide a rational and robust framework for further exploring the application of anisotropic MB for ultrasound-enhanced drug delivery and imaging applications.

Boosting the Potential of Chemotherapy in Advanced Breast Cancer Lung Metastasis via Micro-Combinatorial Hydrogel Particles.

Breast cancer cell colonization of the lungs is associated with a dismal prognosis as the distributed nature of the disease and poor permeability of the metastatic foci challenge the therapeutic efficacy of small molecules, antibodies, and nanomedicines. Taking advantage of the unique physiology of the pulmonary circulation, here, micro-combinatorial hydrogel particles (µCGP) are realized via soft lithographic techniques to enhance the specific delivery of a cocktail of cytotoxic nanoparticles to metastatic foci. By cross-linking short poly(ethylene glycol) (PEG) chains with erodible linkers within a shape-defining template, a deformable and biodegradable polymeric skeleton is realized and loaded with a variety of therapeutic and imaging agents, including docetaxel-nanoparticles. In a model of advanced breast cancer lung metastasis, µCGP amplified the colocalization of docetaxel-nanoparticles with pulmonary metastatic foci, prolonged the retention of chemotoxic molecules at the diseased site, suppressed lesion growth, and boosted survival beyond 20 weeks post nodule engraftment. The flexible design and modular architecture of µCGP would allow the efficient deployment of complex combination therapies in other vascular districts too, possibly addressing metastatic diseases of different origins.

Augmented efficacy of nano-formulated docetaxel plus curcumin in orthotopic models of neuroblastoma.

Neuroblastoma is a biologically heterogeneous extracranial tumor, derived from the sympathetic nervous system, that affects most often the pediatric population. Therapeutic strategies relying on aggressive chemotherapy, surgery, radiotherapy, and immunotherapy have a negative outcome in advanced or recurrent disease. Here, spherical polymeric nanomedicines (SPN) are engineered to co-deliver a potent combination therapy, including the cytotoxic docetaxel (DTXL) and the natural wide-spectrum anti-inflammatory curcumin (CURC). Using an oil-in-water emulsion/solvent evaporation technique, four SPN configurations were engineered depending on the therapeutic payload and characterized for their physico-chemical and pharmacological properties. All SPN configurations presented a hydrodynamic diameter of ∼ 185 nm with a narrow size distribution. A biphasic release profile was observed for all the configurations, with almost 90 % of the total drug mass released within the first 24 h. SPN cytotoxic potential was assessed on a panel of human neuroblastoma cells, returning IC50 values in the order of 1 nM at 72 h and documenting a strong synergism between CURC and DTXL. Therapeutic efficacy was tested in a clinically relevant orthotopic model of neuroblastoma, following the injection of SH-SY5Y-Luc+ cells in the left adrenal gland of athymic mice. Although ∼ 2 % of the injected SPN per mass tissue reached the tumor, the overall survival of mice treated with CURC/DTXL-SPN was extended by 50 % and 25 % as compared to the untreated control and the monotherapies, respectively. In conclusion, these results demonstrate that the therapeutic potential of the DTXL/CURC combination can be fully exploited only by reformulating these two compounds into systemically injectable nanoparticles.

Microfluidic assembly of "Turtle-Like" shaped solid lipid nanoparticles for lysozyme delivery.

After two decades of research in the field of nanomedicine, nanoscale delivery systems for biologicals are becoming clinically relevant tools. Microfluidic-based fabrication processes are replacing conventional techniques based on precipitation, emulsion, and homogenization. Here, the focus is on solid lipid nanoparticles (SLNs) for the encapsulation and delivery of lysozyme (LZ) as a model biologic. A thorough analysis was conducted to compare conventional versus microfluidic-based production techniques, using a 3D-printed device. The efficiency of the microfluidic technique in producing LZ-loaded SLNs (LZ SLNs) was demonstrated: LZ SLNs were found to have a lower size (158.05 ± 4.86 nm vs 180.21 ± 7.46 nm) and higher encapsulation efficacy (70.15 ± 1.65 % vs 53.58 ± 1.13 %) as compared to particles obtained with conventional methods. Cryo-EM studies highlighted a peculiar turtle-like structure on the surface of LZ SLNs. In vitro studies demonstrated that LZ SLNs were suitable to achieve a sustained release over time (7 days). Enzymatic activity of LZ entrapped into SLNs was challenged on Micrococcus lysodeikticus cultures, confirming the stability and potency of the biologic. This systematic analysis demonstrates that microfluidic production of SLNs can be efficiently used for encapsulation and delivery of complex biological molecules.

Efficacy of molecular and nano-therapies on brain tumor models in microfluidic devices.

The three-dimensional (3D) organization of cells affects their mobility, proliferation, and overall response to treatment. Spheroids, organoids, and microfluidic chips are used in cancer research to reproduce in vitro the complex and dynamic malignant microenvironment. Herein, single- and double-channel microfluidic devices are used to mimic the spatial organization of brain tumors and investigate the therapeutic efficacy of molecular and nano anti-cancer agents. Human glioblastoma multiforme (U87-MG) cells were cultured into a Matrigel matrix embedded within the microfluidic devices and exposed to different doses of free docetaxel (DTXL), docetaxel-loaded spherical polymeric nanoparticles (DTXL-SPN), and the aromatic N-glucoside N-(fluorenylmethoxycarbonyl)-glucosamine-6-phosphate (Fmoc-Glc6P). We observed that in the single-channel microfluidic device, brain tumor cells are more susceptible to DTXL treatment as compared to conventional cell monolayers (50-fold lower IC50 values). In the double-channel device, the cytotoxicity of free DTXL and DTXL-SPN is comparable, but significantly lowered as compared to the single-channel configuration. Finally, the administration of 500 µM Fmoc-Glc6P in the double-channel microfluidic device shows a 50 % U87-MG cell survival after only 24 h, and no deleterious effect on human astrocytes over 72 h. Concluding, the proposed microfluidic chips can be used to reproduce the 3D complex spatial arrangement of solid tumors and to assess the anti-cancer efficacy of therapeutic compounds administrated in situ or systemically.

Sustained inhibition of CC-chemokine receptor-2 via intraarticular deposition of polymeric microplates in post-traumatic osteoarthritis.

Posttraumatic osteoarthritis (PTOA) is mostly treated via corticosteroid administration, and total joint arthroplasty continues to be the sole effective intervention in severe conditions. To assess the therapeutic potential of CCR2 targeting in PTOA, we used biodegradable microplates (µPLs) to achieve a slow and sustained intraarticular release of the CCR2 inhibitor RS504393 into injured knees and followed joint damage during disease progression. RS504393-loaded µPLs (RS-µPLs) were fabricated via a template-replica molding technique. A mixture of poly(lactic-co-glycolic acid) (PLGA) and RS504393 was deposited into 20 × 10 µm (length × height) wells in a polyvinyl alcohol (PVA) square-patterned template. After physicochemical and toxicological characterizations, the RS504393 release profile from µPL was assessed in PBS buffer. C57BL/6 J male mice were subjected to destabilization of the medial meniscus (DMM)/sham surgery, and RS-µPLs (1 mg/kg) were administered intraarticularly 1 week postsurgery. Administrations were repeated at 4 and 7 weeks post-DMM. Drug free-µPLs (DF-µPLs) and saline injections were performed as controls. Mice were euthanized at 4 and 10 weeks post-DMM, corresponding to the early and severe PTOA stages, respectively. Knees were evaluated for cartilage structure score (ACS, H&E), matrix loss (safranin O score), osteophyte formation and maturation from cartilage to bone (cartilage quantification), and subchondral plate thickness. The RS-µPL architecture ensured the sustained release of CCR2 inhibitors over several weeks, with ~ 20% of RS504393 still available at 21 days. This prolonged release improved cartilage structure and reduced bone damage and synovial hyperplasia at both PTOA stages. Extracellular matrix loss was also attenuated, although with less efficacy. The results indicate that local sustained delivery is needed to optimize CCR2-targeted therapies.

A Coarse-Grained Molecular Dynamics Description of Docetaxel-Conjugate Release from PLGA Matrices.

Despite the extensive use of poly-lactic-glycolic-acid (PLGA) in biomedical applications, computational research on the mesoscopic characterization of PLGA-based delivery systems is limited. In this study, a computational model for PLGA is proposed, developed, and validated for the reproducibility of transport properties that can influence drug release, the rate of which remains difficult to control. For computational efficiency, coarse-grained (CG) models of the molecular components under consideration were built using the MARTINI force field version 2.2. The translocation free energy barrier ΔGt* across the PLGA matrix in the aqueous phase of docetaxel and derivatives of varying sizes and solubilities was predicted via molecular dynamics (MD) simulations and compared with experimental release data. The thermodynamic quantity ΔGt* anticipates and can help explain the release kinetics of hydrophobic compounds from the PLGA matrix, albeit within the limit of a drug concentration below a critical aggregation concentration. The proposed computational framework would allow one to predict the pharmacological behavior of polymeric implants loaded with a variety of payloads under different conditions, limiting the experimental workload and associated costs.

Management of osteoarthritis: From drug molecules to nano/micromedicines.

With the change in lifestyle and aging of the population, osteoarthritis (OA) is emerging as a major medical burden globally. OA is a chronic inflammatory and degenerative disease initially manifesting with joint pain and eventually leading to permanent disability. To date, there are no drugs available for the definitive treatment of osteoarthritis and most therapies have been palliative in nature by alleviating symptoms rather than curing the disease. This coupled with the vague understanding of the early symptoms and methods of diagnosis so that the disease continues as a global problem and calls for concerted research efforts. A cascade of events regulates the onset and progression of osteoarthritis starting with the production of proinflammatory cytokines, including interleukin (IL)-1ß, IL-6, tumor necrosis factor (TNF)-α; catabolic enzymes, such as matrix metalloproteinases (MMPs)-1, -3, and -13, culminating into cartilage breakdown, loss of lubrication, pain, and inability to load the joint. Although intra-articular injections of small and macromolecules are often prescribed to alleviate symptoms, low residence times within the synovial cavity severely impair their efficacy. This review will briefly describe the factors dictating the onset and progression of the disease, present the current clinically approved methods for its treatment and diagnosis, and finally elaborate on the main challenges and opportunities for the application of nano/micromedicines in the treatment of osteoarthritis. Thus, future treatment regimens will benefit from simultaneous consideration of the mechanobiological, the inflammatory, and tissue degradation aspects of the disease. This article is categorized under: Nanotechnology Approaches to Biology > Nanoscale Systems in Biology Implantable Materials and Surgical Technologies > Nanotechnology in Tissue Repair and Replacement.