Comparative Analysis of PEG-Free and PEG-Based Self-Emulsifying Drug Delivery Systems for Enhanced Oral Bioavailability of Therapeutic (Poly) Peptides.

This study aims to compare the potential of Polyethylene glycol (PEG-free and PEG-based self-emulsifying drug delivery systems (SEDDS) for the oral administration of insulin glargine (IG). Hydrophobic ion pairs (HIPs) of IG are formed using various counterions. HIPs are assessed for log P octanol/water and dissociation behavior. They are incorporated into SEDDS based on polyglycerol (PG) and zwitterionic surfactant (ZW) using response surface methodology and compared to conventional PEG-SEDDS in size, stability, and log D SEDDS/release medium. Oral IG bioavailability in PG/ZW-SEDDS and PEG-SEDDS is evaluated in rats. Among the various counterions studied, IG-BIS (bis(isotridecyl)sulfosuccinate) HIPs demonstrated the highest log P and an improved dissociation profile. PG/ZW-SEDDS and PEG-SEDDS have similar ≈40 nm sizes and are stable over 24 h. Both formulations have log D > 4 in water and >2 in 50 mM phosphate buffer pH 6.8. PG/ZW-SEDDS yielded an oral bioavailability of 2.13 ± 0.66% for IG, while the employment of PEG-SEDDS resulted in an oral bioavailability of 1.15 ± 0.35%. This study highlights the prospective utilization of PEG-free SEDDS involving the concurrent application of PG and ZW surfactants, an alternative to conventional PEG surfactants, for improved oral therapeutic (poly) peptide delivery.

Selecting Counterions to Improve Ionized Hydrophilic Drug Encapsulation in Polymeric Nanoparticles.

Hydrophobic ion pairing (HIP) can successfully increase the drug loading and control the release kinetics of ionizable hydrophilic drugs, addressing challenges that prevent these molecules from reaching the clinic. Nevertheless, polymeric nanoparticle (PNP) formulation development requires trial-and-error experimentation to meet the target product profile, which is laborious and costly. Herein, we design a preformulation framework (solid-state screening, computational approach, and solubility in PNP-forming emulsion) to understand counterion-drug-polymer interactions and accelerate the PNP formulation development for HIP systems. The HIP interactions between a small hydrophilic molecule, AZD2811, and counterions with different molecular structures were investigated. Cyclic counterions formed amorphous ion pairs with AZD2811; the 0.7 pamoic acid/1.0 AZD2811 complex had the highest glass transition temperature (Tg; 162 °C) and the greatest drug loading (22%) and remained as phase-separated amorphous nanosized domains inside the polymer matrix. Palmitic acid (linear counterion) showed negligible interactions with AZD2811 (crystalline-free drug/counterion forms), leading to a significantly lower drug loading despite having similar log P and pKa with pamoic acid. Computational calculations illustrated that cyclic counterions interact more strongly with AZD2811 than linear counterions through dispersive interactions (offset π-π interactions). Solubility data indicated that the pamoic acid/AZD2811 complex has a lower organic phase solubility than AZD2811-free base; hence, it may be expected to precipitate more rapidly in the nanodroplets, thus increasing drug loading. Our work provides a generalizable preformulation framework, complementing traditional performance-indicating parameters, to identify optimal counterions rapidly and accelerate the development of hydrophilic drug PNP formulations while achieving high drug loading without laborious trial-and-error experimentation.

Preparation and characterisation of self-emulsifying drug delivery system (SEDDS) for enhancing oral bioavailability of metformin hydrochloride using hydrophobic ion pairing complexation.

AIM: The aim of this study was preparation of a self-emulsifying drug delivery system (SEEDS) containing metformin hydrochloride. METHODS: Hydrophobic ion paired complexes were prepared by electrostatic interaction between metformin and sodium lauryl sulphate (SLS). The nanodroplets were optimised using two-level full factorial methodology and their morphology were examined. In vitro release of metformin from SEDDS was evaluated in simulated gastric and intestinal fluids. Finally, the ex-vivo efficacy of the optimised formulation in enhancing the intestinal permeability of metformin was evaluated using non-everted intestinal sac. RESULTS: The data revealed that in weight ratio 1:4(metformin: SLS), the highest recovery was achieved. The physico-chemical properties of the optimised nano-droplets including size, polydispersity index (PdI), zeta potential, and loading efficiency (%) were 192.33 ± 9.9 nm, 0.275 ± 0.051; -1.52 mV, and 93.75 ± 0.77% (w/w), respectively. CONCLUSIONS: The data obtained from the intestinal transport study demonstrated that SEDDS can significantly enhance the oral permeability of the compound.

Chemistry and Geometry of Counterions Used in Hydrophobic Ion Pairing Control Internal Liquid Crystal Phase Behavior and Thereby Drug Release.

The combination of Flash NanoPrecipitation and hydrophobic ion pairing (HIP) is a valuable approach for generating nanocarrier formulations of ionic water-soluble drugs with controllable release properties dictated by liquid crystalline structuring of the ion pairs. However, there are few examples of this in practice in the literature. This work aims to decipher the influence of the nature of the hydrophobic counterion used in HIP and its consequent impact on liquid crystalline structuring and drug release. The hypothesis of this study was that hydrophobic counterions with different head and tail groups used for FNP with HIP would give rise to different liquid crystalline structures, which in turn would result in different drug release behavior. A cationic, water-soluble antibiotic, polymixin B, was complexed with eight different hydrophobic counterions with varying head and tail groups and encapsulated into nanocarriers 100-400 nm in size prepared using FNP. Sixteen formulations were assessed for internal structure by synchrotron small-angle X-ray scattering, and drug release was measured in vitro in physiological conditions. The liquid crystalline phases formed depended on the counterion head group and tail geometry, drug:counterion charge ratio, and the ionic strength and pH of the release medium. Drug release occurred more rapidly when no liquid crystalline phases were present and more slowly when higher-ordered phases existed. Specific findings include that phosphonic acid counterions lead to the formation of lamellar structures that persisted at pH 2.0 but were not present at pH 7.3. In contrast, sulfonic acids lead to lamellar or hexagonal phases that persisted at both pH 7.3 and 2.0, while hydrophobic counterions without alkyl tails did not form internal structures. It was also clear that the lipophilicity of the counterion does not dictate drug release. These findings confirm that the liquid crystalline phase behavior of the drug:counterion complex dictates drug release and significantly improves our understanding of the types of controlled release formulations that are possible using FNP with HIP.

Hydrophobic ion-pairing assembled liposomal Rhein with efficient loading for acute pancreatitis treatment.

AIM: The present study aimed to develop liposomal Rhein by employing a hydrophobic ion-pairing technique (HIP) for improved pancreatitis therapy. METHODS: F127 modified liposomal Rhein (F127-RPC-Lip) was prepared using a two-step process consisting of complexation first, followed by a film-ultrasonic dispersion step. The drug-phospholipid interaction was characterised by FT-IR and P-XRD. Particle size and morphology were investigated using DLS and TEM, respectively. Biodistribution and therapeutic efficacy of F127-RPC-Lip were evaluated in a rat model of acute pancreatitis. RESULTS: F127-RPC-Lip achieved efficient drug encapsulation after complexation with lipids through non-covalent interactions and had an average hydrodynamic diameter of about 141 nm. F127-RPC-Lip demonstrated slower drug release (55.90 ± 3.60%, w/w) than Rhein solution (90.27 ± 5.11%) within 24 h. Compared with Rhein, F127-RPC-Lip exhibited prolonged systemic circulation time, superior drug distribution, and attenuated injury in the pancreas of rats post-injection. CONCLUSIONS: HIP-assembled liposomes are a promising strategy for Rhein in treating pancreatitis.

Imaging-Guided Delivery of a Hydrophilic Drug to Eukaryotic Cells Based on Its Hydrophobic Ion Pairing with Poly(hexamethylene guanidine) in a Maleated Chitosan Carrier.

Imaging-guided delivery is developed for hydrophobic drugs, and to a much lesser extent, hydrophilic ones. In this work we have designed a novel strategy for real-time monitoring of hydrophilic drug delivery. Traditionally, the drug and the dye are covalently attached to a nanocarrier or are electrostatically adsorbed. Recently, we found an efficient way to bind the drug by ion-paring with an appropriate counter-ion to form the aggregate that embeds a hydrophobic dye with a considerable fluorescence enhancement. We synthesized a series of carbocyanine dyes of hydrophobicity sufficient for solubilization in hydrophobic ion pairs, which restores their emission in the near-infrared (NIR) region upon the formation of the ternary aggregates. To avoid using toxic surfactants, we applied an amphiphilic polymer-oligomer poly(hexamethylene guanidine) (PHMG) as a counter-ion. Сeftriaxone was used as a model hydrophilic drug ensuring the highest fluorescent signal. The so-formed drug-counter-ion-dye aggregates were encapsulated into a cross-linked maleated chitosan carrier. Confocal laser scanning microscopy (CLSM) studies have demonstrated internalization of the encapsulated model drug by breast adenocarcinoma cells at 40 min after treatment. These results suggest the potential application of hydrophobic ion pairs containing an NIR dye in imaging-guided delivery of hydrophilic compounds.

Permeation-Enhancing Nanoparticle Formulation to Enable Oral Absorption of Enoxaparin.

This study tests the hypothesis that association complexes formed between enoxaparin and cetyltrimethylammonium bromide (CTAB) augment permeation across the gastrointestinal mucosa due to improved encapsulation of this hydrophilic macromolecule within biocompatible poly (lactide-co-glycolide, PLGA RG 503) nanoparticles. When compared with free enoxaparin, association with CTAB increased drug encapsulation efficiency within PLGA nanoparticles from 40.3 ± 3.4 to 99.1 ± 1.0%. Drug release from enoxaparin/CTAB PLGA nanoparticles was assessed in HBSS, pH 7.4 and FASSIFV2, pH 6.5, suggesting effective protection of PLGA-encapsulated enoxaparin from unfavorable intestinal conditions. The stability of the enoxaparin/CTAB ion pair complex was pH-dependent, resulting in more rapid dissociation under simulated plasma conditions (i.e., pH 7.4) than in the presence of a mild acidic gastrointestinal environment (i.e., pH 6.5). The intestinal flux of enoxaparin complexes across in vitro Caco-2 cell monolayers was greater when encapsulated within PLGA nanoparticles. Limited changes in transepithelial transport of PLGA-encapsulated enoxaparin complexes in the presence of increasing CTAB concentrations suggest a significant contribution of size-dependent passive diffusion as the predominant transport mechanism facilitating intestinal absorption. Graphical abstract.

Spray drying OZ439 nanoparticles to form stable, water-dispersible powders for oral malaria therapy.

BACKGROUND: OZ439 is a new chemical entity which is active against drug-resistant malaria and shows potential as a single-dose cure. However, development of an oral formulation with desired exposure has proved problematic, as OZ439 is poorly soluble (BCS Class II drug). In order to be feasible for low and middle income countries (LMICs), any process to create or formulate such a therapeutic must be inexpensive at scale, and the resulting formulation must survive without refrigeration even in hot, humid climates. We here demonstrate the scalability and stability of a nanoparticle (NP) formulation of OZ439. Previously, we applied a combination of hydrophobic ion pairing and Flash NanoPrecipitation (FNP) to formulate OZ439 NPs 150 nm in diameter using the inexpensive stabilizer hydroxypropyl methylcellulose acetate succinate (HPMCAS). Lyophilization was used to process the NPs into a dry form, and the powder's in vitro solubilization was over tenfold higher than unprocessed OZ439. METHODS: In this study, we optimize our previous formulation using a large-scale multi-inlet vortex mixer (MIVM). Spray drying is a more scalable and less expensive operation than lyophilization and is, therefore, optimized to produce dry powders. The spray dried powders are then subjected to a series of accelerated aging stability trials at high temperature and humidity conditions. RESULTS: The spray dried OZ439 powder's dissolution kinetics are superior to those of lyophilized NPs. The powder's OZ439 solubilization profile remains constant after 1 month in uncapped vials in an oven at 50 °C and 75% RH, and for 6 months in capped vials at 40 °C and 75% RH. In fasted-state intestinal fluid, spray dried NPs achieved 80-85% OZ439 dissolution, to a concentration of 430 µg/mL, within 3 h. In fed-state intestinal fluid, 95-100% OZ439 dissolution is achieved within 1 h, to a concentration of 535 µg/mL. X-ray powder diffraction and differential scanning calorimetry profiles similarly remain constant over these periods. CONCLUSIONS: The combined nanofabrication and drying process described herein, which utilizes two continuous unit operations that can be operated at scale, is an important step toward an industrially-relevant method of formulating the antimalarial OZ439 into a single-dose oral form with good stability against humidity and temperature.

Hydrophobic Ion Pairing of Peptide Antibiotics for Processing into Controlled Release Nanocarrier Formulations.

Nanoprecipitation of active pharmaceutical ingredients (APIs) to form nanocarriers (NCs) is an attractive method of producing formulations with improved stability and biological efficacies. However, nanoprecipitation techniques have not been demonstrated for highly soluble peptide therapeutics. We here present a model and technique to encapsulate highly water-soluble biologic APIs by manipulating API salt forms. APIs are ion paired with hydrophobic counterions to produce new API salts that exhibit altered solubilities suitable for nanoprecipitation processing. The governing rules of ion pair identity and processing conditions required for successful encapsulation are experimentally determined and assessed with theoretical models. Successful NC formation for the antibiotic polymyxin B requires hydrophobicity of the ion pair acid to be greater than logP = 2 for strong acids and greater than logP = 8 for weak acids. Oleic acid with a logP = 8, and pKa = 5, appears to be a prime candidate as an ion pair agent since it is biocompatible and forms excellent ion pair complexes. NC formation from preformed, organic soluble ion pairs is compared to in situ ion pairs where NCs are made in a single precipitation step. NC properties, such as stability and release rates, can be tuned by varying ion pair molecular structure and ion pair-to-API molar ratios. For polymyxin B, NCs ≈ 100-200 nm in size, displaying API release rates over 3 days, were produced. This work demonstrates a new approach that enables the formation of nanoparticles from previously intractable compounds.

Development and in vitro characterization of self-emulsifying drug delivery system (SEDDS) for oral opioid peptide delivery.

AIM: In this study, self-emulsifying drug delivery system (SEDDS) for oral delivery of opioid peptide dalargin were developed and characterized in vitro. METHODS: Dalargin lipophilicity was increased by O-esterification of tyrosine OH group, hydrophobic ion pairing, or a combination thereof. Distribution coefficients (log D) of lipidized dalargin derivatives were determined. Then, dalargin was incorporated in chosen SEDDS, namely SEDDS-1, composed of 50% Capmul 907, 40% Cremophor EL, and 10% propylene glycol and comparatively more lipophilic SEDDS-2 composed of 30% Captex 8000, 30% Capmul MCM, 30% Cremophor EL, and 10% propylene glycol. Additionally, SEDDS were characterized regarding droplet size, polydispersity index (PDI), cloudy point, physical stability and stability against pancreatic lipase. Furthermore, mucus permeating properties of SEDDS and their ability to protect the incorporated dalargin against proteolysis by trypsin, α-chymotrypsin, elastase, simulated gastric fluid (SGF), and simulated intestinal fluid (SIF) were evaluated. RESULTS: The highest dalargin drug payload of 4.57% in SEDDS-2 was achieved when dalargin palmitate (pDAL) was ion paired with sodium dodecyl sulfate (SDS) in molar ratio 1:1. Moreover, SEDDS-1 and SEDDS-2 had a narrow droplet size distribution with average droplet sizes of 42.1 and 33.1 nm with PDI of 0.042 and 0.034, respectively. Lipolysis study showed that within 30 min 78.5% of SEDDS-1 and 92.1% of SEDDS-2 were digested. In addition, both SEDDS exhibited mucus permeating properties as well as a protective effect against enzymatic degradation by trypsin, α-chymotrypsin, elastase, SGF and SIF. CONCLUSION: The results of this study suggest that the developed SEDDS could be considered for oral opioid peptide delivery.

Preparation and characterization of self nano-emulsifying drug delivery system (SNEDDS) for oral delivery of heparin using hydrophobic complexation by cationic polymer of ß-cyclodextrin.

OBJECTIVE: The aim of this study was the preparation of a self nano-emulsifying drug delivery system (SNEDDS) for oral delivery of heparin. SIGNIFICANCE: Preparation of hydrophobic complexes between heparin as the hydrophilic macromolecule and cationic polymer of ß-cyclodextrin (CPßCD) was considered for preparation of orally administered SNEDDS in which the drug incorporated in internal oil phase of O/W nano-droplets. METHODS: Hydrophobic complexes of heparin-CPßCD were prepared by electrostatic interaction. The lipophilic feature of complexes was characterized by determining their partition co-efficients. SNEDDS prototypes were prepared by mixing liquid paraffin, Tween 80, propylene glycol and ethanol, diluted 1:100 in an aqueous medium. Central composite response surface methodology was applied for statistical optimization. Independent variables were the amount of liquid paraffin and the amount of Tween 80, while responses were size and poly dispersity index (PdI). Optimized SNEDDS were studied morphologically using transmission electron microscopy (TEM). In vitro release of heparin was studied in the simulated gastric and simulated intestinal media. RESULTS: The data revealed that in molar ratio 1:3 (heparin:CPßCD), the n-octanol recovery was maximized and reached 67.6 ± 11.86%. Size, PdI, zeta potential, EE% in gastric medium and EE% in intestinal medium for optimized nano-droplets were reported as 307 ± 30.51 nm, 0.236 ± 0.02, +2.1 ± 0.66 mV, 90.2 ± 0.04 and 96.1 ± 0.73%, respectively. Microscopic images revealed spherical nano-droplets. The obtained data revealed no burst release of heparin from nano-droplets. CONCLUSIONS: The obtained results indicate that SNEDDS could be regarded as a good candidate for oral delivery of heparin as the hydrophilic macromolecule.

Oral self-nanoemulsifying peptide drug delivery systems: impact of lipase on drug release.

It was the aim of this study to evaluate the impact of lipases on the release behaviour of a peptide drug from oral self-nanoemulsifying drug delivery systems. Octreotide was ion paired with the anionic surfactants deoxycholate, decanoate, oleate and dodecylsulphate. The lipophilic character of these complexes was characterised by determining the n-octanol/buffer pH 7.4 partition coefficient. In the following the most hydrophilic complex was incorporated in a likely lipase degradable self-nanoemulsifying drug delivery systems (SNEDDS) formulation containing a triglyceride (olive oil; Pharm.Eur.) and in a likely not lipase degradable SNEDDS containing lipids and surfactants without any ester bonds. After 1:100 dilutions in artificial intestinal fluid (AIF), the lipid droplets were characterised regarding size distribution. With these SNEDDS, drug release studies were performed in AIF with and without lipase. Results showed that the most hydrophobic complex can be formed with deoxycholate in an octreotide:anionic surfactant ratio of 1:5. Even 73.1 ± 8.1% of it could be quantified in the n-octanol phase. SNEDDS containing octreotide | olive oil | cremophor EL | propylene glycol (2|57|38|3) and octreotide | liquid paraffin | Brij 35 | propylene glycol | ethanol (2|66.5|25|5|1.5) showed after dilution in AIF, a mean droplet size of 232 ± 53 nm and 235 ± 50 nm, respectively. Drug release studies showed a sustained release of octreotide out of these formulations for at least 24 h, whereas > 80% of the drug was released within 2 h in the presence of lipase in the case of the triglyceride containing SNEEDS. In contrast the release profile from ester-free SNEDDS was not significantly altered (p < 0.05) due to the addition of lipase providing evidence for the stability of this formulation towards lipases. According to these results, SNEDDS could be identified as a useful tool for sustained oral peptide delivery taking an enzymatic degradation by intestinal lipases into considerations.

A novel approach for antibody nanocarriers development through hydrophobic ion-pairing complexation.

IgG-Fab fragment, a model antibody protein was hydrophobically modified by a novel approach of ion-pairing complexation. Three different sulphated ion-pairing agents were utilised including sodium dodecyl sulphate, taurocholic acid and dextran sulphate (DS). The formations of hydrophobic ion-pairing (HIP) complexes were dependant on pH and molar ratio of ion-pairing agent to Fab. Aqueous solubilities of HIP complexes were very low compared to Fab alone. In particular, when dextran sulphate was added as ion-pairing agent, formed Fab:DS HIP complexes were least soluble in water. Further, nanoparticles (NPs) loaded with drug and Fab:DS HIP complex were prepared and characterised with respect to encapsulation efficiency and size. We observed significant improvement in encapsulation efficiency for Fab:DS HIP complex-loaded nanoparticles. This study demonstrates a novel approach of formulating antibody-loaded nanoparticles which can also be employed for delivery of large antibodies.

Variations in the inner core affect the pharmacokinetics of indomethacin-encapsulated polymeric micelles.

Hydrophobic ion pairing (HIP) is a drug encapsulation technology that uses electrostatic interactions between a drug and an additive. However, although polymeric micelles can encapsulate hydrophobic drugs in the core, the encapsulated drug often leaks. Therefore, we designed polymeric micelles with HIP functionalized in a hydrophobic inner core using three diblock copolymers comprising polypeptides with different ratios of polar and hydrophobic amino acids and polyethylene glycol (PEG) to encapsulate indomethacin (IND). The three IND-encapsulated HIP micelles showed different area under the curve (AUC) values as an index of blood retention after intravenous injection in mice. Despite having the same PEG shell, IND-PEG-poly(H/F)n showed a 1.56-fold higher AUC than IND-PEG-poly(D/F)n. PEG interface morphologies were evaluated to determine the differences in pharmacokinetic parameters caused by changes in inner core HIP patterns. The micellarized diblock copolymer was desorbed from IND-PEG-poly(D/F)n due to electrostatic repulsion between IND and the diblock copolymer comprising aspartic acid. Our results suggest that changes in the HIP patterns of the micelle inner core affected the PEG interface morphologies, such as PEG density and diblock copolymer desorption from micelles. These phenomena might lead to changes in the interaction of plasma proteins and drug dispositions.

Construction of injectable micron-sized polymorphic vesicles for prolonged local anesthesia with weekly sustained release of ropivacaine.

Currently, to overcome the short half-life of the local anesthetic ropivacaine, drug delivery systems such as nanoparticles and liposomes have been used to prolong the analgesic effect, but they are prone to abrupt release from the site of administration or have poor slow-release effects, which increases the risk of cardiotoxicity. In this study, injectable lipid suspensions based on ropivacaine-docusate sodium hydrophobic ion pairing (HIP) were designed to significantly prolong the duration of analgesia. The resulting ion-paired lipid suspension (HIP/LIPO) had a micrometer scale and a high zeta potential, which facilitates stable in situ retention. The strong interaction between docusate sodium and ropivacaine was verified using thermal and spectroscopic analyses, and the formation of micron-sized polymorphic vesicles was attributed to the mutual stabilizing interactions between ropivacaine-docusate sodium HIP, docusate sodium and lecithin. The HIP/LIPO delivery system could maintain drug release for more than 5 days in vitro and achieve high analgesic efficacy for more than 10 days in vivo, reducing the side effects associated with high drug doses. The stable HIP/LIPO delivery system is a promising strategy that offers a clinically beneficial alternative for postoperative pain management and other diseases.

MCT Nanoemulsions for the Efficient Delivery of siRNA.

In this study, an oil-in-water (o/w) nanoemulsion is used to deliver siRNA targeting Twist1, a protein that contributes to tumor metastasis in a variety of cancers. The FDA-approved oil, medium chain triglycerides (MCT), is used as the hydrophobic phase for the nanoemulsion. The siRNA is paired with dioleoyl-3-trimethylammonium-propane (DOTAP) to form a hydrophobic salt that is soluble at high concentrations in MCT. The resulting MCT/siRNA-DOTAP solution is formulated into a nanoemulsion with an average particle size of 140 nm. The nanoemulsion displays long term stability over the course of 195 days. In an in vivo murine tumor model, the nanoemulsion facilitates a 46% decrease in Twist1 mRNA after 48 h.

QbD Approach-Based Preparation and Optimization of Hydrophobic Ion-Pairing Complex of Lysozyme with Sodium Dodecyl Sulphate to Enhance Stability in Lipid-Based Carriers.

Hydrophobic ion pairing (HIP) complexation was found to be an efficient approach in modulating the release and enhancing the stability and encapsulation of hydrophilic macromolecules such as proteins in hydrophobic nano/microcarriers. The present work strives to develop and optimize the preparation of the HIP complex of the antimicrobial enzyme lysozyme (LYZ) with the ion-pairing agent (IPA) sodium dodecyl sulphate (SDS) relying on the quality-by-design (QbD) approach. The quality target product profile (QTPP) includes the achievement of maximal lipophilicity in a reversible manner to enable the maintenance of biological activity. The related critical quality attributes (CQAs) were defined as complexation efficacy, complex stability, enzyme recovery and activity. Three risk assessment (RA) tools were used to identify and rank the critical process parameters (CPPs) and critical material attributes (CMAs). From this assessment, the pH of the medium, LYZ:SDS molar ratio and drying conditions were determined as high-risk factors that need to be investigated. To the best of our knowledge, for the first time, electrostatic titration was used as a smart approach to determine the optimum molar ratio at different pH values. Based on the predefined CQAs, pH 8 with an LYZ/SDS molar ratio of 1:8 was found to be the optimal condition for complexation efficiency and recovery (%) of a biologically active enzyme. A cost-effective drying process based on a ventilated oven was developed, which resulted in complex qualities comparable to those obtained by the commonly used freeze-drying method. In a nutshell, the optimum conditions for the preparation of the LYZ/SDS HIP complex were efficiently facilitated by the rational application of QbD principles and the utilization of efficient electrostatic titration and ventilated oven-drying methods.

Histidine-based ionizable cationic surfactants: novel biodegradable agents for hydrophilic macromolecular drug delivery.

The aim of this study was to design surfactants based on histidine (His) for hydrophobic ion-pairing and evaluate their safety and efficacy. Lauryl, palmitoyl and oleyl alcohol, as well as 2-hexyl-1-decanol were converted into surfactants with histidine as head-group via esterification. The synthesized His-surfactants were characterized regarding pKa, critical micellar concentration (CMC), biodegradability, toxicity on Caco-2 cells, and ability to provide endosomal escape. Furthermore, the suitability of these agents to be employed as counterions in hydrophobic ion pairing was evaluated. Chemical structures were confirmed by 1H-NMR, FT-IR, and MS. The synthesized surfactants showed pKa values ranging from 4.9 to 6.0 and CMC values in the range of 0.3 to 7.0 mM. Their biodegradability was proven by enzymatic cleavage within 24 h. Below the CMC, His-surfactants did not show cytotoxic effects on Caco-2 cells (cell viability > 80%). All His-surfactants showed the ability to provide endosomal escape in a pH-dependent manner in the range of 5.2 to 6.8. Complexes formed between His-surfactants and heparin or plasmid DNA (pDNA) via hydrophobic ion pairing showed at least 100-fold higher lipophilicity than the correspondent model drugs. According to these results, His-surfactants might be a promising safe tool for delivering hydrophilic macromolecular drugs and nucleic acids.

Nor-LAAM loaded PLGA microparticles for treating opioid use disorder.

The treatment landscape for opioid use disorder (OUD) faces challenges stemming from the limited efficacy of existing medications, poor adherence to prescribed regimens, and a heightened risk of fatal overdose post-treatment cessation. Therefore, there is a pressing need for innovative therapeutic strategies that enhance the effectiveness of interventions and the overall well-being of individuals with OUD. This study explored the therapeutic potential of nor-Levo-α-acetylmethadol (nor-LAAM) to treat OUD. We developed sustained release nor-LAAM-loaded poly (lactic-co-glycolic acid) (PLGA) microparticles (MP) using a hydrophobic ion pairing (HIP) approach. The nor-LAAM-MP prepared using HIP with pamoic acid had high drug loading and exhibited minimal initial burst release and sustained release. The nor-LAAM-MP was further optimized for desirable particle size, drug loading, and release kinetics. The lead nor-LAAM-MP (F4) had a relatively high drug loading (11 wt%) and an average diameter (19 µm) and maintained a sustained drug release for 4 weeks. A single subcutaneous injection of nor-LAAM-MP (F4) provided detectable nor-LAAM levels in rabbit plasma for at least 15 days. We further evaluated the therapeutic efficacy of nor-LAAM-MP (F4) in a well-established fentanyl-addiction rat model, and revealed a marked reduction in fentanyl choice and withdrawal symptoms in fentanyl-dependent rats. These findings provide insights into further developing long-acting nor-LAAM-MP for treating OUD. It has the potential to offer a new effective medication to the existing sparse armamentarium of products available to treat OUD.

Hydrophobic ion pairing and microfluidic nanoprecipitation enable efficient nanoformulation of a small molecule indolamine 2, 3-dioxygenase inhibitor immunotherapeutic.

Blockade of programmed cell death-1 (PD-1) is a transformative immunotherapy. However, only a fraction of patients benefit, and there is a critical need for broad-spectrum checkpoint inhibition approaches that both enhance the recruitment of cytotoxic immune cells in cold tumors and target resistance pathways. Indoleamine 2, 3-dioxygenase (IDO) small molecule inhibitors are promising but suboptimal tumor bioavailability and dose-limiting toxicity have limited therapeutic benefits in clinical trials. This study reports on a nanoformulation of the IDO inhibitor navoximod within polymeric nanoparticles prepared using a high-throughput microfluidic mixing device. Hydrophobic ion pairing addresses the challenging physicochemical properties of navoximod, yielding remarkably high loading (>10%). The nanoformulation efficiently inhibits IDO and, in synergy with PD-1 antibodies improves the anti-cancer cytotoxicity of T-cells, in vitro and in vivo. This study provides new insight into the IDO and PD-1 inhibitors synergy and validates hydrophobic ion pairing as a simple and clinically scalable formulation approach.