Controlled Release Capability Research Articles

Synthesis of layered organic–inorganic nanohybrid zinc–aluminium-2-(4-chlorophenoxy)-2-methyl propionic acid with controlled release properties

A plant growth regulator, 2-(4-chlorophenoxy)-2-methyl propionic acid (CFA), was successfully intercalated into zinc–aluminium layered double hydroxide (ZAL) forming a new nanohybrid composite, zinc–aluminium-2-(4-chlorophenoxy)-2-methylpropionate layered double hydroxide (ZAC). Well-crystallized nanohybrid composite was obtained when the material was synthesized with zinc to aluminium molar ratio, R = 4 and 0.2 M CFA. Due to the intercalation of CFA, basal spacing expanded from 8.9 A in the ZAL to 21.4 A in the ZAC. The FTIR spectra of the ZAC nanohybrid composite show resemblance peaks of the ZAL and CFA indicating the inclusion of the organic compound into the LDH interlamellae. The loading percentage of CFA is 40.0% (w/w) calculated based on the percentage of carbon in the sample. The BET surface area increased from 1.0 to 70.0 m2 g−1 due to the inclusion of CFA into the ZAL interlamellae and associated expansion of the layered structure. Release of CFA from the ZAL interlayer was found to be dependent on the affinity of the incoming anion, in the order of phosphate > sulfate > nitrate with the percentage saturated release of 74, 54, and 42%, respectively. This study indicates the potential application of zinc–aluminium-layered double hydroxide as a host for plant growth regulator, CFA, with controlled release capability.

Overview of Studies Regarding Mesoporous Silica Nanomaterials and Their Biomedical Application.

Synthesis of mesoporous silica nanomaterials occurred in the late 1980s and early 1990s. Since then, we have seen dramatic increase in the number of publication dealing with this nanomaterial. In particular, there have been a large number of publications regarding biomedical application of mesoporous silica nanoparticles including their use in cancer therapy and diagnosis. Various surface modification strategies have been explored to enhance drug loading and to target them to tumor. In addition, the nanoparticles have been armed with controlled release capability. In this review, we point out some crucial issues regarding the material, which will be discussed in more detail in chapters of this volume.

Bio-derived cellulose nanofibril reinforced poly(N-isopropylacrylamide)-g-guar gum nanocomposite: An avant-garde biomaterial as a transdermal membrane

The delivery of diltiazem hydrochloride in therapeutical doses has attracted an immense research interest. However, its slower penetration through the transdermal route has stipulated to develop a competent transdermal membrane. Therefore, a nanocomposite based patch was formulated by exploring co-polymer and jute derived nano-cellulose. Poly(N-isopropylacrylamide) was grafted into guar gum (GG-g-PNIPAAm) with different feeding ratios. The co-polymer formation was authenticated by FTIR and 13C NMR spectra. The nanocomposite were prepared by incorporating nanofibre (0.5–2 wt%) into GG-g-PNIPAAm. The structural and morphological studies supported good interactions and presence of nano-cellulose on co-polymer. GG-g-PNIPAAm has showed higher thermostability than guar gum. Moreover, the addition of CNF has improved the thermo-mechanical and barrier properties of the nanocomposite. The nanocomposite containing 1 wt% CNF was found to be best performing. The patch showed in-vitro cyto-compatibility and non-irritant behaviour. The in-vitro release study of best nanocomposite revealed controlled drug release capability with 7.78 and 22.9% after 5 and 20 h, respectively.

Microsphere-Based Scaffolds in Regenerative Engineering.

Microspheres have long been used in drug delivery applications because of their controlled release capabilities. They have increasingly served as the fundamental building block for fabricating scaffolds for regenerative engineering because of their ability to provide a porous network, offer high-resolution control over spatial organization, and deliver growth factors/drugs and/or nanophase materials. Because they provide physicochemical gradients via spatiotemporal release of bioactive factors and nanophase ceramics, microspheres are a desirable tool for engineering complex tissues and biological interfaces. In this review we describe various methods for microsphere fabrication and sintering, and elucidate how these methods influence both micro- and macroscopic scaffold properties, with a special focus on the nature of sintering. Furthermore, we review key applications of microsphere-based scaffolds in regenerating various tissues. We hope to inspire researchers to join a growing community of investigators using microspheres as tissue engineering scaffolds so that their full potential in regenerative engineering may be realized.

Conditional Network Assembly and Targeted Protein Retention via Environmentally Responsive, Engineered β-Roll Peptides.

Stimulus-responsive biomaterials have applications in many areas of biotechnology, such as tissue engineering, drug delivery, and bioelectrocatalysis. The intrinsically disordered repeat-in-toxin (RTX) domain is a conformationally dynamic peptide that gains β-roll secondary structure when bound to calcium ions. A smart hydrogel platform was constructed by genetically fusing two rationally designed mutant RTX domains: first, a mutant peptide with hydrophobic interfaces capable of calcium-dependent network assembly, and second, another mutant that conditionally binds the model target protein lysozyme. In this way, the calcium-induced control over the secondary structure of the β-roll peptide was exploited to regulate both the cross-linking and lysozyme-binding functionalities. The constructed biomaterial exhibited calcium-dependent gelation and target molecule retention, and erosion experiments showed that β-roll peptides with a higher affinity for lysozyme produced more robust hydrogel networks. This work demonstrates the use of RTX domains for introducing two useful features simultaneously, network cross-linking and target protein binding, and that the calcium-dependent regulation of these systems can be useful for controlling bulk self-assembly and controlled release capabilities.

Multifunctional and Redox-Responsive Self-Assembled Magnetic Nanovectors for Protein Delivery and Dual-Modal Imaging.

Nanoparticle (NP) based model carriers present an emerging strategy for protein delivery. However, constructing a multifunctional nanocarrier with high loading capacity, diagnostic imaging capacity, and controlled release capability is a tremendous challenge for protein delivery systems. Thus, we herein report on the fabrication of redox-responsive magnetic nanovectors (termed RMNs) through self- assembly of Fe3O4 NPs and redox-responsive polymer ligands, which could effectively transport protein and trigger intracellular protein release. These RMNs also exhibited low toxicity, high stability, biocompatibility, and T2-weighted contrast-enhancement properties. In addition, they presented a quantized positively charged surface that had the capacity to load cyanine 5.5 (Cy5.5) labeled human serum albumin (HSA) with high loading efficiency (∼84%) via electrostatic interactions and which favored cellular uptake. Notably, studies of the in vitro protein release showed that HSA-Cy5.5-loaded RMNs (RMNs-HSA-Cy5.5) presented minimal cumulative release behavior under physiological conditions but release was rapidly enhanced under high glutathione concentration conditions. Confocal microscopy further revealed that protein was delivered and localized at the perinuclear region of tumor cells. Moreover, the in vivo imaging results confirmed that RMNs-HSA-Cy5.5 could serve as a dual-modal probe for simultaneous near-infrared fluorescence (NIRF) imaging and magnetic resonance (MR) imaging, which can be used for breast cancer diagnosis, and verified higher tumor accumulation of transported protein in a living body. Overall, we believe that these multifunctional RMNs exhibit great promise for protein delivery, cancer diagnosis and therapy, and multimodal imaging, as well as clinical applications.

Fe-Ptナノ粒子とヒドロキシプロピルセルロースからなる熱応答性磁気キャリアの作製

The present paper describes a fabrication of magnetic composite of Fe-Pt nanoparticles and hydroxylpropyl cellulose (HPC) for magnetically guided drug delivery system with a capability of thermo-responsive controlled release. The composites are fabricated by a synthesis of Fe-Pt nanoparticles in a reaction solution containing HPC coated silica particles; Fe-Pt nanoparticles are grown on HPC deposited on the silica particles. The island-like composites of HPC and Fe-Pt NPs are observed to be densely deposited on the silica particles by transmission electron microscopy. Thermal responsiveness is evaluated by temperature-dependence of particle size distribution using dynamic light scattering; the thermal responsiveness is successfully confirmed even after the thermal history up to 533 K during synthesis.

Modulating antioxidant activity and the controlled release capability of laccase mediated catechin grafting of chitosan

Abstract Laccase from ascomycete Myceliophthora thermophila was used for the oxidation and grafting of catechin (CA) and chitosan (CS). The enzymatic grafting of CA into CS resulted in the improvement of natural properties of chitosan films. The successful grafting of CA to CS was supported by UV–vis and Fourier transform infrared (FT-IR) spectroscopies. The differences in the molecular weight and the degree of deacetylation of the CS affected on the enzymatic grafting rate and thus on the film properties like swelling behaviour, erosion rate, thermal property, tensile strength and breaking strain. The CS films prepared by enzymatic grafting presented high antioxidant activity compared to native CS films. The controlled release profiles of methylene blue and coomassie brilliant blue dye, having different acidity/basicity properties, loaded films were also studied with different release medium. The types of molecule loaded into films, the preparation manner of films and the release medium were decisive parameters affecting the release rate.

Superparamagnetic Reduction/pH/Temperature Multistimuli-Responsive Nanoparticles for Targeted and Controlled Antitumor Drug Delivery

Multistimuli-responsive polymeric nanoparticles with core-shell architecture were prepared by coating superparamagnetic Fe3O4 nanoparticle cores with reduction/pH dual-responsive poly(methacrylic acid) (PMAA) as shells and thermal-responsive poly(N-isopropylacrylamide) (PNIPAM) as a "gatekeeper" on the surface via two-stage distillation precipitation polymerization. The Fe3O4@PMAA nanoparticles were synthesized using N,N-bis(acryloyl)cystamine (BACy) as cross-linker which would be easily biodegradable in the presence of dithiothreitol (DTT) or glutathione (GSH). The cumulative release profile was investigated under different conditions, such as media pH, reductive agents, and temperature, with doxorubicin hydrochloride (DOX) as a model anticancer drug. They showed a low leakage of DOX at pH 7.4 (less than 11% in 24 h), while the release significantly accelerated at pH 5.0 and 10 mM GSH (over 60% in 5 h), realizing the "triggered release" of drug in the targeted tissues. The nanoparticles exhibited excellent biocompatibility while the DOX-loaded nanoparticles showed great promise of antitumor efficacy as free DOX by the MTT assay and CLSM analysis. The results suggest that the novel biodegradable nanoparticles with high drug loading capacity and multiresponsive controlled release capability could serve as an excellent gene/drug delivery system candidate for cancer therapy.

Nanoformulation of Geranylgeranyltransferase-I Inhibitors for Cancer Therapy: Liposomal Encapsulation and pH-Dependent Delivery to Cancer Cells.

Small molecule inhibitors against protein geranylgeranyltransferase-I such as P61A6 have been shown to inhibit proliferation of a variety of human cancer cells and exhibit antitumor activity in mouse models. Development of these inhibitors could be dramatically accelerated by conferring tumor targeting and controlled release capability. As a first step towards this goal, we have encapsulated P61A6 into a new type of liposomes that open and release cargos only under low pH condition. These low pH-release type liposomes were prepared by adjusting the ratio of two types of phospholipid derivatives. Loading of geranylgeranyltransferase-I inhibitor (GGTI) generated liposomes with average diameter of 50–100 nm. GGTI release in solution was sharply dependent on pH values, only showing release at pH lower than 6. Release of cargos in a pH-dependent manner inside the cell was demonstrated by the use of a proton pump inhibitor Bafilomycin A1 that Increased lysosomal pH and inhibited the release of a dye carried in the pH-liposome. Delivery of GGTI to human pancreatic cancer cells was demonstrated by the inhibition of protein geranylgeranylation inside the cell and this effect was blocked by Bafilomycin A1. In addition, GGTI delivered by pH-liposomes induced proliferation inhibition, G1 cell cycle arrest that is associated with the expression of cell cycle regulator p21CIP1/WAF1. Proliferation inhibition was also observed with various lung cancer cell lines. Availability of nanoformulated GGTI opens up the possibility to combine with other types of inhibitors. To demonstrate this point, we combined the liposomal-GGTI with farnesyltransferase inhibitor (FTI) to inhibit K-Ras signaling in pancreatic cancer cells. Our results show that the activated K-Ras signaling in these cells can be effectively inhibited and that synergistic effect of the two drugs is observed. Our results suggest a new direction in the use of GGTI for cancer therapy.

Preparation and functional characterization of tumor-targeted folic acid-chitosan conjugated nanoparticles loaded with mitoxantrone

Folic acid conjugated chitosan was prepared by cross-linking reaction with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), and then used as a template to prepare folic acid-chitosan (FA–CS) conjugated nanoparticles and load mitoxantrone nanoparticles (FA-CSNP/MTX). Drug dissolution testing, CCK-8 method, and confocal microscopy were used to detect their controlled-release capability in different situations and the specific uptake by HONE1 cells. The experimental results show that the nanoparticles have uniform size distribution of 48–58 nm. The highest encapsulation rate of the particles on mitoxantrone hydrochloride (MTX) is (77.5±1.9)%, and the drug loading efficiency is (18.4±0.4)%. The sustained release effect, cell growth inhibition activity and targeting effect of the FA-CS/MTX nanoparticles are good in artificial gastric fluid and intestinal fluid. It is demonstrated that the FA-CSNP system is a potentially useful system for the targeted delivery of anticancer drug MTX.

Preparation and functional characterization of tumor-targeted folic acid–chitosan conjugate nanoparticles loaded with mitoxantrone

Folic acid conjugated chitosan was prepared by cross-linking reaction with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), and then used as a template to prepare folic acid-chitosan (FA–CS) conjugated nanoparticles and load mitoxantrone nanoparticles (FA-CSNP/MTX). Drug dissolution testing, CCK-8 method, and confocal microscopy were used to detect their controlled-release capability in different situations and the specific uptake by HONE1 cells. The experimental results show that the nanoparticles have uniform size distribution of 48–58 nm. The highest encapsulation rate of the particles on mitoxantrone hydrochloride (MTX) is (77.5±1.9)%, and the drug loading efficiency is (18.4±0.4)%. The sustained release effect, cell growth inhibition activity and targeting effect of the FA-CS/MTX nanoparticles are good in artificial gastric fluid and intestinal fluid. It is demonstrated that the FA-CSNP system is a potentially useful system for the targeted delivery of anticancer drug MTX.

Behavior of POP-calcium carbonate hydrogel as bone substitute with controlled release capability: a study in rat.

Gypsum or calcium sulfate (CS) or plaster of Paris (POP) is considered as a fast degradable material that usually resorbs before the bone defect area is completely filled by new bone. In this study, the incorporation of CaCO3 hydrogel into POP in different compositions was proposed to enhance the bone biological activity of POP and to decrease its degradability. The mechanical and degradation properties of the various materials were characterized by in vitro analysis. Subsequently, the materials were inserted into cylindrically sized bone defects as created into the femoral condyle of rats and left in situ for 1, 4, and 8 weeks. Histological analysis of the retrieved specimens indicated that the addition of CaCO3 hydrogel into POP increased bone formation, angiogenesis and collagen density and resulted into faster bone formation and maturation. It was also confirmed that the degradation rate of the POP decreased by the addition of CaCO3 hydrogel. The in vivo findings did corroborate with the in vitro analysis. In conclusion, the incorporation of CaCO3 hydrogel provides a promising technology to improve the properties of POP, the oldest biomaterial used for bone grafting.

Spatiotemporally controlled delivery of biomolecules (1053.5)

Plasmonic nanoparticles are appealing for biomolecule and drug delivery due to their unique photophysical properties that provide spatiotemporally controlled release capabilities. Emerging techniques using light to control cellular functions, such as in optogenetics, motivates the design of novel methods to achieve mechanical manipulation of the cell. Here we demonstrate the release of a recombinant green fluorescent protein fused with a poly‐histidine tag to facilitate attachment to ~ 40 nm hollow gold nanoshells by nickel‐nitrilotriacetic acid affinity as well as a C‐terminal targeting peptide to promote cellular uptake of the nanoparticle‐protein composite. Laser release of protein was characterized by fluorescence for both particle and cellular studies, which was found to be power and time dependent. The construct presented may serve as a platform for further studies of protein delivery using nanoparticles.Grant Funding Source: This work was supported by National Institutes of Health (NIH) grant R01 EB012637.

Layered Double Hydroxide as Carrier of Herbicide, 2-Methyl-4-Chlorophenoxy Acetic Acid: Physicochemical Characterization and Controlled Release Properties

A new organic-inorganic hybrid nanocomposite Zn-Al-MCPA layered double hydroxide was prepared by intercalation of 2-methyl-4-chlorophenoxy acetic acid (MCPA), into Zn-Al-layered double hydroxide (ZAL) at various concentration of MCPA ranging from 0.1 to 0.7. The pH of the formulation was kept constant at 7.5. Well-ordered hybrid nanocomposite was obtained with 0.4 M MCPA with an expansion of basal spacing from 8.9 A in the ZAL s resemblance peaks of to 19.7 A in the resulting nanocomposite. The FTIR spectra of the nanocomposite shows resemblance peaks of the MCPA and Zn-Al-layered double hydroxide indicates the inclusion of MCPA into the layered double hydroxide with loading of MCPA found to be 45% (w/w). The release of MCPA into various aqueous solution was found to be dependent on the anion in the aqueous solution, in the order of phosphate > sulphate > nitrate with the percentage release of 80%, 44% and 8%, respectively. This study shows that Zn-Al-layered double hydroxide can be used as a host carrier for herbicide, MCPA with controlled release capability.

MedinGel™: a controlled-release platform for intraarticular delivery of osteoarthritis drugs

Purpose: Current osteoarthritis treatments have clear delivery limitations and are principally designed to treat symptoms. Drawbacks include inefficient penetration of drugs into the joint space, systemic side effects and short duration of action. Drugs administered for systemic release have poor synovial penetration that is required to provide relief from OA-related pain and inflammation. High plasma concentrations of many NSAID compounds lead to severe side effects such as stomach pain and ulcers. And in the case of orally administered celecoxib, warnings of increased risk for heart attack and stroke have been issued. MedinCell will present our company's experience in designing polymer-based delivery technology to circumvent these problems with the goal of improving osteoarthritis treatments. We will discuss the use of our MedinGel™ platform for multi-month local delivery of small molecules and biologics to help control pain, reduce inflammation and promote cartilage repair. Without controlled release capability, drugs injected directly into the joint spaces are often cleared within hours. Yet daily intraarticular injections are untenable, as they would have to be administered by trained clinicians and would severely impact the patients’ quality of life. Additionally, a high frequency of injections into the joint could have increased risk of bacterial infection. By tailoring formulations to specific drugs for a range of delivery durations, MedinCell is confident that intraarticular formulations can deliver a sustained therapeutic drug concentration over multiple months to obviate current concerns and improve both treatment efficacy and patient compliance. Methods: After evaluation of drug solubility and set up of in vitro-release assays, a series of sets of formulations evaluating combinations of polymers at different concentrations were prepared. The MedinGel matrix is composed of mixtures of drug molecules with solubilized, bioresorbable polymers. Within minutes after injection through a fine needle, the aqueous environment of the joint leads to a phase-separation of the formulated polymers and formation of a three-dimensional matrix. This resulting semi-solid hydrogel depot entraps and protects drug molecules within the joint space, and minimizes clearance to the plasma. Results: Candidate formulations that provided a range of drug release rates in vivo were evaluated. The best candidates were then tested by intraarticular injection. MedinCell has designed intraarticular formulations of OA drug therapies that maintained therapeutic synovial concentrations for a 3-month duration in animals. Conclusions: The MedinGel platform enables control over the initial drug burst characteristics, allowing the remainder of the drug to be released over a predetermined duration via polymer hydrolysis and drug diffusion until the depot is completely resorbed. No covalent modification of the active substance is required, which can simplify regulatory and development pathways. We will present data showing differential compartmentalization of drug between joint and plasma when comparing normal injection to MedinGel delivery. Additionally, MedinCell expects that this technology will provide effective delivery of disease-modifying caspase inhibitors and proteins (such as BMP7) for trauma-induced arthritis.

The release behavior and kinetic evaluation of tramadol HCl from chemically cross linked Ter polymeric hydrogels.

Background and the purpose of the studyHydrogels, being stimuli responsive are considered to be effective for targeted and sustained drug delivery. The main purpose for this work was to study the release behavior and kinetic evaluation of Tramadol HCl from chemically cross linked ter polymeric hydrogels.MethodsTer-polymers of methacrylate, vinyl acetate and acrylic acid cross linked with ethylene glycol dimethacrylate (EGDMA) were prepared by free radical polymerization. The drug release rates, dynamic swelling behavior and pH sensitivity of hydrogels ranging in composition from 1-10 mol% EGDMA were studied. Tramadol HCl was used as model drug substance. The release behavior was investigated at pH 8 where all formulations exhibited non-Fickian diffusion mechanism.Results and major conclusionAbsorbency was found to be more than 99% indicating good drug loading capability of these hydrogels towards the selected drug substance. Formulations designed with increasing amounts of EGDMA had a decreased equilibrium media content as well as media penetrating velocity and thus exhibited a slower drug release rate. Fitting of release data to different kinetic models indicate that the kinetic order shifts from the first to zero order as the concentration of drug was increased in the medium, showing gradual independency of drug release towards its concentration. Formulations with low drug content showed best fitness with Higuchi model whereas those with higher concentration of drug followed Hixson-Crowell model with better correlation values indicating that the drug release from these formulations depends more on change in surface area and diameter of tablets than that on concentration of the drug. Release exponent (n) derived from Korse-Meyer Peppas equation implied that the release of Tramadol HCl from these formulations was generally non-Fickian (n > 0.5 > 1) showing swelling controlled mechanism. The mechanical strength and controlled release capability of the systems indicate that these co-polymeric hydrogels have a great potential to be used as colon drug delivery device through oral administration.

Synthesis of nanocomposite 2-methyl-4-chlorophenoxyacetic acid with layered double hydroxide: physicochemical characterization and controlled release properties

A new organic–inorganic hybrid nanocomposite Zn–Al-MCPA-layered double hydroxide (ZAM) was prepared by intercalation of 2-methyl-4-chlorophenoxyacetic acid (MCPA) into Zn–Al-layered double hydroxide (ZAL) at various concentration of MCPA ranging from 0.1 to 0.7 M. The pH of the synthesis was kept constant at 7.5. Well-ordered hybrid nanocomposite was obtained with 0.4 M MCPA with an expansion of basal spacing from 8.9 A in the ZAL to 19.7 A in the resulting nanocomposite. The FTIR spectra of the nanocomposite show resemblance peaks of the MCPA and Zn–Al-layered double hydroxide indicating the inclusion of MCPA into the layered double hydroxide. The average particle size of ZAL and ZAM in this study was 115 and 128 nm, respectively. Percentage loading of MCPA was found to be 45.0 % (w/w), calculated based on the percentage of carbon in the sample. The release of MCPA into various aqueous solution was found to be dependent to the anion in the aqueous solution in the order of phosphate > sulfate > chloride with the percentage release of 80, 44, and 8 %, respectively. This study shows that Zn–Al-layered double hydroxide can be used as a host carrier for herbicide, MCPA, with controlled release capability.

Single Emulsion-Solvent Evaporation Technique and Modifications for the Preparation of Pharmaceutical Polymeric Nanoparticles

In recent years, there has been an increased interest in using nanoparticles for drug delivery and pharmaceutical development. Nanoparticles can offer significant advantages over the conventional drug delivery systems in terms of high drug loading, stability and specificity, controlled release capability, and the ability to deliver both hydrophilic and hydrophobic drug molecules through various routes of administration. This review article focuses on the use of the single emulsion solvent evaporation method, the first method proposed for the preparation of polymeric nanoparticles, and modifications that have been developed over the years to improve the results obtained with this technique.

Improving Product Quality with Entrapped Stable Emulsions: From Theory to Industrial Application

Entrapment of sub-micron scale emulsions containing active ingredients into macro-scale matrices has exhibited great potential as a delivery vehicle with controlled release capabilities, however optimization remains unrealized. Reported here are methods used to improve product quality by optimizing the emulsion formation steps. These methods are in conjunction with the precepts of Process Intensification (PI). Success with pharmaceutics and chemical reacting systems provides a strategy for a wide range of applications; the emphasis here being nutraceutics. Use of a nano-technology platform assists in: (a) product quality improvements through better nutrient dispersion, and thus bio-efficacy; and (b) production efficiencies through implementation of PI concepts. A continuous methodology, utilizing these PI concepts, that approximates a bottom-up approach to the creation of sub-micron and nano-emulsions is the basis of the technology presented here. Note that solid particles may result during post-processing. The metrics of successful processing include obtainment of nano-scale species with minimal input energy, reduced processing steps at higher throughput rates, and improved quality without over-usage of key ingredients. In addition to flavor and wellness characteristics, product stability for extended shelf life along with an appreciable cargo load in the entrapped emulsion is a major concern. Experimental protocols and path forward recommendations to overcome challenges and meet expectations in these emerging opportunities are also presented.