The successful development of siRNA-lipid nanoparticle (LNP) therapeutics depends heavily on the consistent quality of excipients, particularly the ionizable lipid MC3, which is commonly used in clinically approved products such as Onpattro. MC3 plays a key role in RNA encapsulation, endosomal escape, and biocompatibility. As regulatory frameworks evolve to support the development of generic siRNA-LNPs, this work presents the first systematic comparison of how vendor-dependent variations in the purity of MC3 affect the physicochemical properties and in vitro performance of siRNA-LNPs. A multi-platform analytical workflow for impurity analysis revealed that the dienone byproduct of MC3 was the most abundant impurity across all three vendors tested and it was retained in the LNP formulation. Despite variations in purity levels (86-99%) and modest differences in particle size, polydispersity index (PDI), and distinct thermal behavior, the LNPs displayed similar critical quality attributes (CQAs), including encapsulation efficiency, surface charge, apparent pKₐ, and internal structure. In vitro studies showed that all LNPs achieved approximately 60% transthyretin (TTR) mRNA and protein knockdown in HepG2 cells at a 5 nM dose, and maintained HepG2 cell viability at 30 nM, indicating comparable efficacy and biocompatibility. Human peripheral blood mononuclear cell (PBMC) assays showed no significant cytokine induction for LNPs formulated with MC3 from any source. In stability assessments, despite no noticeable chemical changes in LNPs stored over 70 days at room temperature, changes in physicochemical properties such as particle size and PDI were observed. These changes, however, did not impact gene silencing efficiency or biocompatibility. Overall, differences in MC3 purity across the evaluated vendors influenced certain physicochemical attributes of siRNA-LNPs, while in vitro performance remained largely unaffected. Controlling lipid impurity profiles, however, is important for ensuring consistent physicochemical characteristics and supporting quality assurance. Collectively, these findings provide valuable insights for manufacturing and regulatory considerations as siRNA-LNPs progress toward generic development.

There is a growing demand for high-concentration protein formulations (HCPFs), driven by certain therapeutics that require high doses and the volume limitations of subcutaneous administration. However, downstream processing and fill-finish for these HCPFs plays a critical role and remains a major limitation. It accounts for nearly 60% of the total production cost and faces significant challenges in scalability and efficiency. These challenges are largely attributed to inherent complexity, which involve multiple unit operations such as ultrafiltration, bulk drug substance freezing, thawing, mixing, sterile filtration and drug product fill-finish. This review provides a comprehensive overview of downstream processing and fill-finish for high-concentration protein formulations, highlighting the key challenges, particularly related to drug stability, (such as protein aggregation, or particulate formation), viscosity and solubility (such as turbidity or phase separation). Addressing these multifactorial challenges demands various strategies that include formulation and process optimization, and the use of specialized delivery systems which are discussed herewith. Moreover, emerging trends such as process analytical technologies (PAT), use of non-aqueous delivery vehicles and potential of using solid suspensions are also highlighted as promising strategies for overcoming the current limitations. Finally, to address the real-world applications, case studies of two FDA-approved high-concentration formulations (up to 200 mg/mL) are presented illustrating the need for formulation and process design for enabling successful clinical translation.

PurposeThe commercial In situ forming implants (ISFIs) use either a dual-syringe system requiring pre-injection syringe mixing, or a pre-mixed ready-to-inject syringe. The purpose of this study is to comprehensively evaluate the impact of these two mixing approaches on ISFIs' properties as well as in vitro performance.MethodsComputed tomography (CT) imaging and scanning electron microscopy were used to access implant structure ad morphology. High-performance liquid chromatography was used for drug release determination. Gel permeation chromatography was used to evaluate polymer degradation. The CT contrast agent iohexol was used as a model compound and was fully dissolved in the matrix regardless of the mixing approach.ResultsSyringe-mixed ISFIs contained air bubbles, while ready-to-inject formulation displayed a more uniform polymer matrix without them. The introduction of air bubbles exhibited slower solvent NMP release, altered drug deposition, increased volume expansion, and enhanced initial degradation rate. CT imaging revealed that ready-to-inject formulation was more uniform, whereas syringe-mixed implants appeared to have more heterogenous cavity feature.ConclusionsThe introduction of air bubbles through pre-injection mixing could potentially impact drug release and ISFI's performance. This study elucidates the comparison between two mixing techniques as a reference in the developmental process of future products.

A poly(lactic-co-glycolic acid) (PLGA)-based in situ-forming implant (ISFI) is a long-acting injectable composed of active ingredients, a biodegradable polymer and a biocompatible solvent. Understanding the role of PLGA end-caps is crucial for designing ISFI formulations due to their impact on drug delivery performance. In this study, using leuprolide acetate as a model drug, we characterized the ISFIs using computed tomography (CT) imaging to explore the influence of PLGA end-cap on the implant's formation and drug-release behavior. CT imaging enabled a detailed characterization of implant morphology and internal structure, providing new insights into the relationship between phase inversion, implant formation, and in vitro release performance. Acid-ended PLGA showed a higher initial burst release with 100% of the solvent released at 9 days and a faster release duration of the drug in the in vitro release profile. In contrast, ester-ended PLGA showed a more prolonged release profile, with the plateau phase not reached until approximately day 50. Morphological analysis from CT images of the in vitro implants revealed that acid-ended PLGA formed spherical implants with a dense outer layer, whereas ester-ended PLGA resulted in irregular shapes with a larger size expansion. In vivo CT imaging confirmed these trends, although implant evolution occurred more rapidly in biological environments. Overall, this research highlights the impact of PLGA end-caps on the performance of ISFI, providing a scientific basis for formulation development, evaluation, and optimization of ISFIs.

More than 70% of patients with ovarian cancer are diagnosed with metastasis, in which tumors spread to the peritoneal cavity. The current standards of care are intravenous (IV) or intraperitoneal (IP) injection of small-molecule anticancer drugs, such as cis-diamminedichloro platinum-(II) (cisplatin). Although IP injection enables delivery of higher drug concentrations to the tumor sites, the small molecules have low retention times. To overcome these challenges, we present two alternative nanoparticle-based formulations, namely, cisplatin-loaded dendritic mesoporous silica nanoparticles (Pt-MSNs) and poly-(acrylic acid) (PAA)-modified dendritic MSNs (Pt-PAA-MSNs). For both formulations, we obtained stable particles with uniform shapes/sizes that did not aggregate 15 days after in vitro drug release. We achieved loading amounts as high as 15% for Pt-MSNs and 18% for Pt-PAA-MSNs. PAA offers additional advantages such as a more sustained cisplatin release and a faster release in an acidic tumor environment due to the pH sensitivity of the polymer. The formulation showed a 4.8-times improvement in the half maximal inhibitory concentration (IC50) against cancer cells. Pharmacokinetic (PK) studies further demonstrated an increased retention time of cisplatin in the peritoneal cavity, as absorbed Pt in the bloodstream reduced from 1.75 μg for free cisplatin (peaked as early as 30 min after IP injection) to 0.05 μg peaked at 300 min for Pt-PAA-MSNs. The biodistribution results in a metastasis-induced mouse model showed an increased accumulation of cisplatin in tumors of MSN-administered animals compared to those that received free cisplatin. These findings demonstrate the potential of Pt-PAA-MSNs as a promising platform for improving the efficacy and retention of cisplatin in peritoneal ovarian cancer treatment.

Lipid nanoparticles (LNPs) have emerged as a versatile delivery platform for improving pharmacokinetic performance, protecting nucleic acid cargo, and enabling tissue- and cell-specific targeting. Continued advancement of LNP-based therapeutics requires a deeper understanding of how raw material quality, formulation parameters, nanoparticle architecture, and biological context collectively influence clinical performance. In this Perspective, we discuss key challenges, practical insights, and lessons learned from ongoing LNP development efforts, with emphasis on characterization strategies, delivery specificity, scale-up considerations, long-term stability, and emerging applications of artificial intelligence. We highlight the importance of rational design principles, robust and reproducible manufacturing practices, comprehensive analytical characterization, and innovative approaches to support the next generation of LNP technologies.

In situ forming implants (ISFIs) are long-acting drug delivery systems that solidify upon administration to enable sustained release of therapeutics. They are administered by a minimally invasive injection, have adjustable release duration up to six-months or longer, enable localized delivery, and are easier to manufacture compared with other modalities of long-acting injectables (LAIs). Despite clinical successes with several FDA-approved products in the last three decades, ISFIs remain challenging to develop due to complexity of the formulation and fundamental understanding of drug release mechanism. This review summarizes the status of ISFI technology and highlights advances in technical understanding with a focus on translation to drug products. It provides the first review on the formation and degradation processes of ISFIs, including the interplay of solvent exchange, polymer attributes, and drug physicochemical properties in shaping burst release, diffusion-driven release, and degradation-mediated release phases. Beyond the discussion of attributes, the morphology and inner structure change along with the implant formation and degradation is first reviewed. We summarize characterization tools-such as electron microscopy, MRI, CT, ultrasound, and spectroscopy-that provide mechanistic insights into implant morphology, microenvironmental changes, and drug deposition. Emphasis is placed on how these mechanistic understanding can inform critical quality attributes, improve in vitro-in vivo correlations, and support the development of drug products. Collectively, the knowledge presented will be instructive to understand the formation and degradation process of ISFI, further facilitating the development of ISFI products.

Abstract Nucleic acid therapeutics have emerged as the third major class of therapeutics, following small molecules and antibodies. Lipid nanoparticles (LNPs) serve as the leading platform for nucleic acid delivery. The mRNA-LNPs showed clinical success in COVID-19 vaccines, but they suffer from ultra-low temperature storage requirement that limits global transportation and distribution. Drying technologies, especially freeze-drying, offer solutions to maintain stability at a higher temperature. This review highlights the intrinsic stability of mRNA-LNPs, the status of using freeze-drying for improving the stability of mRNA-LNPs, the potential challenges during freeze-drying, and the strategies for maintaining the stability of mRNA-LNPs during freeze-drying and storage. Beyond mRNA, we compare drying strategies for other nucleic acid-LNPs (NA-LNPs), including siRNA, saRNA, circRNA, pDNA, miRNA, and the CRISPR system, underscoring the impact of structure on the drying of these NA-LNPs. We also discuss alternative freeze-drying and drying methods, including continuous freeze-drying based on spin-freezing, thin film freeze-drying, spray freeze-drying, spray drying, and alcohol dilution freeze-drying, for improving stability and suitability for different routes of administration. Overall, this review provides insight for the development of a solid form of NA-LNPs with maintained stability and potency.

The peritoneal cavity presents both unique challenges and promising opportunities for targeted therapy in malignancies like ovarian, gastric, pancreatic, and colorectal cancers. Intraperitoneal drug delivery offers significant pharmacokinetic advantages over intravenous administration by achieving high local drug concentrations and tumor-specific delivery potential while minimizing systemic toxicity. Despite these theoretical advantages, the clinical implementation of intraperitoneal therapy is limited by several barriers, including restricted tissue penetration, incomplete peritoneal coverage, rapid drug clearance, catheter-related complications, posttreatment peritoneal adhesions, and ascites-induced permeability dysregulation. This review highlights three advanced strategies developed to overcome these obstacles: (1) particulate-based delivery systems, such as nanoparticles to enhance tumor specificity through passive accumulation, active targeting and on-demand drug release in response to internal or external stimuli; (2) Sustained drug release hydrogels and (3) pressurized intraperitoneal aerosol chemotherapy. Despite promising preclinical and clinical advancements, successful translation requires systematic optimization of multiple parameters, such as ascites dynamics, tumor heterogeneity, and multidrug resistance. The integration of advanced delivery technologies with a comprehensive understanding of peritoneal physiology remains crucial for achieving safe and effective clinical applications.

The presence of drug nanoparticles is believed to promote oral drug absorption through the particle drifting effect. These particles act as vehicles that penetrate the diffusion boundary layer adjacent to the intestinal membrane, releasing the drug near the mucosal surface, and thereby enhancing drug absorption. Despite extensive in vitro studies demonstrating the potential of this mechanism to improve drug absorption, in vivo evaluations of the particle drifting effect remain limited. In this work, we systematically evaluated the particle drifting effect in vivo in both mice and rats using enzalutamide as a model drug. To assess the impact of particle concentration, dose-escalation studies were performed to examine regions where absorption was driven by either solubilized drug or nanoparticles. The formation of amorphous drug nanoparticles was shown to significantly enhance drug absorption, confirming the role of the particle drifting effect. Nonlinear pharmacokinetics were observed in the nanoparticle absorption region, consistent with non-sink dissolution conditions in the boundary layer at high particle concentrations. Additionally, animal species-specific differences were observed, likely due to variations in bile salt concentration in the gastrointestinal fluids of mice and rats. Moreover, in vitro-in vivo relationships were established. These results provide valuable insights for early-stage formulation development, particularly for amorphous solid dispersions, before first-in-human studies where different surrogate tests and animal models are used, and should contribute to a broader understanding of how to predict the oral bioavailability of nanoparticle-containing formulations.

This paper evaluates and compares two viable approaches for on-demand fortification of pea-based snacks using binder jet 3D printing. Thiamine hydrochloride was added to an aqueous binder solution that selectively joins the pea flour layer-by-layer. Three thiamine levels, namely, 0.5, 1, and 1.5 mg, were targeted based on recommended dietary allowances for toddlers, young females, and pregnant females. The final amount of thiamine dosed into the sample depends on the concentration of thiamine in the binder solution and the total amount of thiamine solution dispensed. In the first approach, the amount of solution dispensed was fixed but the thiamine concentration was adjusted for each target dose level. In the second approach, the thiamine concentration in the binder solution was fixed, but the amount of thiamine solution dispensed was varied. Varying amounts of binder solution containing no thiamine was also dispensed to maintain the same level of binder saturation and consequently the mechanical properties. Overall, the second approach had an average dosing accuracy of 94%, which was 3% higher than that of the first approach. In practice, the second approach is also more versatile and preferred as only one thiamine solution is required. For both schemes, the compressive modulus and strength of the baked samples were comparable to those of shortbread, but the amount of thiamine decreased by about 14% after baking, attributed to thermal degradation. The thiamine amount in the baked samples did not change further when vacuum-packed and stored in the dark for one week.

Postsurgical pain management remains a persistent challenge for patients and healthcare providers. This work presents an implantable, degradable drug release platform based on silk biomaterials designed to be placed at a desired surgical site intraoperatively to release the local anesthetic bupivacaine in a sustained manner to prevent and treat postsurgical pain. Methacrylate monomers containing different pendant groups are used to generate brush-like polymers on silk fibroin films by surface-initiated reversible addition-fragmentation chain transfer polymerization. These brushes have side groups to control polymer hydrophilicity and facilitate drug attachment via a hydrolyzable tether. Spectroscopy and contact angle goniometry are used to characterize the chemical composition and hydrophobicity of the synthesized films at each synthetic step. In vitro culture and in vivo implant studies show no differences in biocompatibility compared with unmodified silk films. Bupivacaine can be continually released for at least 7 days. The amount of drug released in vitro is increased by increasing the hydrophilicity of the brush-like polymer, and the released anesthetic is effective at blocking the conduction of action potentials of C-fibers and Aδ-fibers ex vivo. These degradable films show promise as a platform to achieve controllable, continuous delivery of local anesthetics for pain control after surgery.

The mRNA lipid nanoparticle (mRNA-LNP) is a promising platform for vaccines and a variety of therapeutic areas, as demonstrated by the effective mRNA-LNP formulations in COVID-19 vaccines. While in-vitro transfection studies are crucial for optimizing mRNA-LNP formulations, few studies examine how cell line selection and reporter genes affect transfection efficiency. In our study, we investigated the in-vitro transfection efficiency of firefly luciferase mRNA-LNP on Jurkat cells, L-929 cells, and HEK 293 T cells. Jurkat cells, as a suspension cell line, displayed low transfection efficiency. The luciferase expression showed a non-linear relationship with mRNA dose, and cytotoxicity was observed with even low concentrations of mRNA. L-929 cells showed a linear relationship between bioluminescence and mRNA concentration, but only at low levels of mRNA, and their luciferase expression is limited. HEK 293 T cells are superior because of a strong linear dose–response and higher signal intensity. However, when using the luciferase-based assay for mRNA-LNP transfection, we observed high intra-group variations with signal fluctuated among technical replicates of the same formulation. In contrast, eGFP mRNA exhibited high reproducibility for the in-vitro transfection tests (coefficient of variation < 10

Levo-alpha-acetylmethadol (LAAM) has a long duration of action offering numerous behavioral and clinical advantages for treating opioid use disorders. In this study, we aim to develop a novel oral immediate-release capsule of LAAM, as an alternative to methadone, the major medication that requires daily visits. LAAM solid dispersion-coated pellets were developed with VIVAPUR® MCC Spheres 700 as the core, and LAAM-carrier coating solution was sprayed onto the core pellets using a fluid bed coater. The coating solution formulation was optimized for reduced coating time and improved drug loading efficiency. Design of Experiments (DoE) studies were conducted, revealing the impact of critical process parameters-nozzle air pressure and airflow-on the critical quality attributes of the LAAM-coated pellets. Capsules with different dosage strengths were prepared. Capsule weight variation, content uniformity, and in vitro release tests were conducted based on USP guidelines for quality assessment. The LAAM capsules showed the immediate release profile. The storage stability of the LAAM capsules was evaluated under accelerated condition and room temperature for 6 months. The weight gain, drug content, and dissolution profile were evaluated. The results showed that drug contents and immediate-release properties remained after 6 months of storage. Pharmacokinetic (PK) study in rabbits showed that capsules have longer tmax as well as higher Cmax of LAAM compared with oral solution. These capsules have a similar AUC of LAAM and its metabolites to the oral solution. It has been demonstrated that the developed LAAM capsules have the desired properties for subsequent clinical studies.

This investigation compares in vitro release, ex vivo release and permeation, and in vivo ocular pharmacokinetics to render biologically informed evaluations of ophthalmic semi-solid drug products containing dexamethasone (hydrophobic) and tobramycin (hydrophilic). Both drugs were formulated with three petrolatum matrices (IGI® 320 A, IGI® 386, or Spectrum®) with distinct rheological character and benchmarked against the reference listed drug, Tobradex®. Temperature-sweep rheology revealed that IGI® 386 most closely reproduced the viscoelastic profile of the reference product. USP Apparatus I release testing with surfactant-free medium provided maximal discrimination for dexamethasone (Tobradex® > IGI® 320 A > IGI® 386 > Spectrum®), and rank-order release rates correlated strongly with ex vivo corneal permeation and in vivo corneal exposure. In contrast, tobramycin required a polysorbate-containing medium to resolve formulation differences in vitro, yet those differences did not persist ex vivo or in vivo, consistent with its rapid dissolution and diffusion, which attenuate matrix effects. The data demonstrate that drug solubility dictates the choice of biorelevant release conditions in petrolatum-based ophthalmic ointments: surfactant-free media capture formulation-dependent release for hydrophobic actives, whereas hydrophilic actives may yield artifactual discrimination when surfactant is present. However, formulations indistinguishable in vitro were typically similar in their in vivo ocular pharmacokinetics. By integrating tiered models, the framework enhances understanding of critical quality attributes, supports regulatory decision-making, and may help reduce reliance on animal studies, thereby expediting the development of therapeutically equivalent generic ophthalmic ointments.

The present study systematically investigates the impact of active pharmaceutical ingredient (API) variables and oleaginous base characteristics on the in vitro release (IVR) performance of ophthalmic ointments, utilizing dexamethasone as a model drug. The interplay between selected attributes (i.e., particle size distribution, crystallinity, and polymorphic form for API, and rheological factors for compendial-grade white petrolatum) and IVR performance was investigated. APIs from different vendors exhibited variations in crystallinity and polymorphism. Ointments containing amorphous dexamethasone presented higher release amounts/rates compared to crystalline counterparts, emphasizing the role of physical state in release kinetics. Variations in particle size of this lipophilic API (5.4 - 21.2 mu m) did not appear to impact IVR performance significantly. In contrast, white petrolatum 's rheological attributes, which varied substantially within USP-grade petrolatum, were found to critically affect the drug release rate and extent of the ointment. The study 's comprehensive analysis establishes a coherent connection between the quality attributes of both API and petrolatum and IVR, delineating their intricate interdependent effects on ophthalmic ointment performance. These findings provide reference to formulation design, quality control, and regulatory considerations within the pharmaceutical industry, fostering a robust foundational understanding of commonly overlooked quality attributes in ophthalmic ointments.

Advanced ovarian cancer with peritoneal metastasis is challenging to treat. Limited tumor delivery and penetration of the therapeutics to deep tumor regions are significant barriers to effective treatment. The rising radiopharmaceuticals offer hopes for patients through targeted delivery. However, site specific delivery avoiding off target effect remain critical and challenging. We have developed radioactive 166Holmium loaded mesoporous silica nanoparticles (166Ho-MSNs) that exhibited predominant accumulation to peritoneal metastases of ovarian cancer upon intraperitoneal administration. It was observed that fluorescence labeled radioactive 166Ho-MSNs distributed throughout the tumor tissues, while non-radioactive fluorescent 165Ho-MSNs showed mainly tumor surface deposition of MSNs. The deep penetration leads to uniform therapeutic radiation distribution and absorbed doses within tumors as demonstrated by the dosimetry analysis. The radiation dosing regimen consisting of two 100 µCi 166Ho-MSN doses separated by 7 days decreased tumor activity and increased the overall lifespan and ascites free survival in several models of IP tumor-bearing mice. These findings illustrate that 166Ho-MSN is promising for the treatment of ovarian peritoneal metastasis, with selective targeting advantage of the nanoparticles and limited off-target radiation exposure.