Biological and intracellular fates of drug nanocrystals through different delivery routes: Recent development enabled by bioimaging and PK modeling.

Nanocrystals have contributed to exciting improvements in the delivery of poorly water-soluble drugs. The biological and intracellular fates of nanocrystals are currently under debate. Due to the remarkable commercial success in enhancing oral bioavailability, nanocrystals have originally been regarded as a simple formulation approach to enhance dissolution. However, the latest findings from novel bioimaging tools lead to an expanded view. Intact nanocrystals may offer long-term durability in the body and offer drug delivery capabilities like those of other nano-carriers. This review renews the understanding of the biological fates of nanocrystals administered via oral, intravenous, and parenteral (e.g., dermal, ocular, and pulmonary) routes. The intracellular pathways and dissolution kinetics of nanocrystals are explored. Additionally, the future trends for in vitro and in vivo quantification of nanocrystals, as well as factors impacting the biological and intracellular fates of nanocrystals are discussed. In conclusion, nanocrystals present a promising and underexplored therapeutic opportunity with immense potential.

In vivo dissolution of poorly water-soluble drugs: Proof of concept based on fluorescence bioimaging.

In vitro‒in vivo correlation (IVIVC) of solid dosage forms should be established basically between in vitro and in vivo dissolution of active pharmaceutical ingredients. Nevertheless, in vivo dissolution profiles have never been accurately portrayed. The current practice of IVIVC has to resort to in vivo absorption fractions (F a). In this proof-of-concept study, in vivo dissolution of a model poorly water-soluble drug fenofibrate (FNB) was investigated by fluorescence bioimaging. FNB crystals were first labeled by near-infrared fluorophores with aggregation-caused quenching properties. The dyes illuminated FNB crystals but quenched immediately and absolutely once been released into aqueous media, enabling accurate monitoring of residual drug crystals. The linearity established between fluorescence and crystal concentration justified reliable quantification of FNB crystals. In vitro dissolution was first measured following pharmacopoeia monograph protocols with well-documented IVIVC. The synchronicity between fluorescence and in vitro dissolution of FNB supported using fluorescence as a measure for determination of dissolution. In vitro dissolution correlated well with in vivo dissolution, acquired by either live or ex vivo imaging. The newly established IVIVC was further validated by correlating both in vitro and in vivo dissolution with F a obtained from pharmacokinetic data.

The biological fate of orally administered mPEG-PDLLA polymeric micelles.

The biological fate of polymeric micelles (PMs) following oral administration was investigated in this study to better understand the contribution of transport of integral PMs to oral absorption. To track integral PMs, near-infrared fluorophores with aggregation-caused quenching properties were utilized to label PMs comprised of methoxy poly(ethylene glycol)-poly(D,L-lactic acid) (mPEG-PDLLA) copolymers and methoxy poly(ethylene glycol)-distearoyl phosphoethanolamine (DSPE-PEG). The particle size of PMs prepared from mPEG2.5k-PDLLA1.25k, mPEG2.5k-PDLLA2.5k, mPEG5k-PDLLA3k, mPEG5k-PDLLA5k and DSPE-PEG2k was 24.5, 29.5, 34.0, 41.4 and 15.6 nm, respectively. After oral administration by gavage to rats, PMs were retained in the gastrointestinal tract for at least 4 h, and the copolymer block chain lengths did not have significant influence. The emergence of fluorescence in the blood and liver served as direct evidence to support oral absorption of integral PMs. Approximately 1-2% of intact particles were absorbed via the lymphatic pathway, but the total amount of PMs that reach the systemic circulation await further elucidation. Confocal laser scanning microscopy added more evidence to support the penetration of integral PMs into the basolateral tissues of microvilli. Cellular uptake efficiency was about 4-7% in Caco-2 cell lines for all PM groups, but was reduced to 1-3% in Caco-2/HT29-MTX co-culture models due to the hindrance by the mucus layers. Approximately 6-12% of integral PMs were transported across Caco-2/HT29-MTX/Raji monolayers, whereas only approximately one-tenth of that amount was transported across Caco-2 and Caco-2/HT29-MTX monolayers. Differences, but not statistically significant, were observed between PM groups in lymphatic uptake, biodistribution, cellular uptake and trans-monolayer transport, possibly owing to difference in block chain lengths as well as particle size. In conclusion, evidence obtained in this study supports penetration of integral PMs across the enteric epithelia, but the total amount may be limited.

Enhanced transdermal delivery of curcumin nanosuspensions: A mechanistic study based on co-localization of particle and drug signals.

Nanosuspensions have received much attention in enhanced transdermal delivery. However, the corresponding mechanisms have not been clarified. In particular, whether nanosuspensions can directly penetrate across the stratum corneum (SC) and what is the transdermal route for the enhanced penetration. Therefore, curcumin (CUR) was adopted in this study as a model drug, while an aggregation-caused quenching (ACQ) probe was physically embedded in CUR nanosuspensions, i.e., the CUR hybrid nanosuspensions (CUR-HNSs), for bioimaging. The ACQ properties enable identification of intact CUR-HNSs. The co-localization of particle and CUR signals was exploited to outline the translocation profiles of intact nanosuspensions as well as the cargoes. Three sizes of CUR-HNSs are prepared, which are spherical and amorphous. CUR is poor in transdermal transport even in propylene glycol solution, which was enhanced by nanosuspensions. Although 400 nm CUR-HNSs present higher steady state flux than 140 nm and 730 nm ones, the cumulative amount of permeated CUR is yet less than 2% of the applied dose at 12 h. Co-localization of CUR and ACQ probe signals indicates that CUR-HNSs can infiltrate into the SC layer and accumulate in the hair follicles. The intact CUR-HNSs cannot enter into the skin. On the contrary, CUR molecules diffuse into the whole skin tissues following dissolution of CUR-HNSs in the SC and the hair follicles. In conclusion, nanosuspensions are advantageous for transdermal delivery of poorly permeable drugs by filtrate into the SC and accumulate in hair follicles.

Slowing down lipolysis significantly enhances the oral absorption of intact solid lipid nanoparticles.

Only a limited amount of orally administered lipid nanoparticles are absorbed as intact particles due to lipolysis by lipases in the gastrointestinal tract. It is hypothesized that by counteracting lipolysis, more particles will survive gastrointestinal digestion and be absorbed as intact particles. In this study, incorporation of a lipase inhibitor orlistat (OLST), as well as polyethylene glycol (PEG) coating, is employed to slow down the lipolysis using solid lipid nanoparticles (SLNs) as model particles. To explore the in vivo behaviors of the particles, near-infrared fluorescent probes with absolute aggregation-caused quenching (ACQ) properties are used to label and track the unmodified, PEG-coated and OLST-loaded SLNs. The in vitro lipolysis study indicates very fast first-order degradation of unmodified SLNs and significantly decreased degradation of OLST-SLNs. Live imaging reveals the same trend of slowed-down lipolysis in vivo which correlates well with the in vitro lipolysis. The scanning of ex vivo gastrointestinal segments confirms the considerably prolonged residence time of OLST-SLNs, mirroring the significantly decreased lipolysis rate. The observation of fluorescence in the blood, though very weak, and in the liver speaks of the oral absorption of intact SLNs. The substantially higher hepatic levels of OLST-SLNs than unmodified SLNs should be attributed to the significantly enhanced survival rate because both particles exhibit similar cellular recognition as well as similar physicochemical properties except for the survival rate. Similarly, slowing down lipolysis also contributes to the significantly enhanced cumulative lymphatic transport of OLST-SLNs (7.56% vs. 1.27% for the unmodified SLNs). The PEG coating slows down the lipolysis rate as well but not to the degree as done by OLST. As a result, the gastrointestinal residence time of PEG-SLNs has been moderately prolonged and the hepatic levels moderately increased. The weakened cellular recognition of PEG-SLNs implies that the enhanced oral absorption is solely ascribed to the slowed-down lipolysis and enhanced mucus penetration. In conclusion, the oral absorption of intact SLNs can be significantly enhanced by slowing down lipolysis, especially by OLST, showing potential as carriers for the oral delivery of labile biomacromolecules.

Hybrid drug nanocrystals.

Nanocrystals show promise to deliver poorly water-soluble drugs to yield systemic exposure. However, our knowledge regarding the in vivo fate of nanocrystals is in its infancy, as nanocrystallization is simply viewed as an approach to enhance the dissolution of drug crystals. The dying crystal phenomenon inspired the development of hybrid nanocrystals by physically embedding fluorophores into the crystal lattice. This approach achieved concurrent therapy and bioimaging and is well-established to study pharmacokinetics and nanocrystal dissolution in vivo. Nanocrystals also offer the advantage of long-term durability in the body for interacting with biological tissues and cells. This review introduces the hybrid nanocrystal technique, including the theoretical concepts, preparation, and applications. We also discuss the latest development in self-discriminative hybrid nanocrystals utilizing environment-responsive probes. This review will stimulate further development and application of nanocrystal-based drug delivery systems for theranostic strategies.

Bioimaging of Intact Polycaprolactone Nanoparticles Using Aggregation-Caused Quenching Probes: Size-Dependent Translocation via Oral Delivery.

The limited information on biological fate impedes the development of more efficient polymeric nanoparticles for oral delivery of bio-macromolecules. In this study, the in vivo fate as well as the trans-epithelia transport of polycaprolactone (PCL) nanoparticles is explored by labeling with aggregation-caused quenching probes, which is capable of identifying intact nanoparticles. Live imaging and confocal laser scan microscopy confirm size-dependent absorption of PCL nanoparticles. In general, reducing particle size favors a faster and more oral absorption. Nanoparticles larger than 200 nm, such as 600 and 2000 nm, cannot be efficiently transported across the intestinal membrane. The absorbed nanoparticles (50 and 200 nm) mainly accumulate in the liver. Lymph may be the main absorption route for PCL nanoparticles, transporting 2.39 ± 1.81% and 0.98 ± 0.58% of administered 50 and 200 nm nanoparticles, respectively. Cellular uptake and transportation of PCL nanoparticles are also size dependent. Both enterocytes and M cells mediated transcytosis are involved in the transport of 50 nm PCL nanoparticles, while the M cell pathway is dominative for other nanoparticles. In conclusion, the study provides a valuable tool for bioimaging of intact polymeric nanoparticles as well as solid evidence supporting size-dependent translocation of the nanoparticles via oral delivery.

Visual validation of the measurement of entrapment efficiency of drug nanocarriers.

Entrapment efficiency (EE) is a crucial parameter for the evaluation of nanocarriers. The accurate measurement of EE demands clear separation of nanocarriers from free drugs, which so far has not been clearly validated due to a lack of functional tools to identify nanocarriers. Herein, an environment-responsive water-quenching fluorophore was employed to label and identify model nanocarriers, polycaprolactone nanoparticles (PN), methoxy polyethylene glycol-poly(d,l-lactic acid) polymeric micelles (PM) and solid lipid nanoparticles (SLN). The separation process of three commonly used methods (centrifugation, ultrafiltration and gel permeation chromatography) was visualized by live imaging. The separation efficiency of the centrifugation method is very poor, especially for PM (40 nm), SLN (100 nm) and PN (100 nm); only PN (200 nm) can be efficiently separated but at a consumption of enormous energy. The ultrafiltration method shows the best separation efficiency with only 0.32-0.93% of leakage of the nanocarriers. Gel permeation chromatography exhibits good separation as well but suffers from low recovery, a potential factor that might compromise the accuracy of EE measurement. In conclusion, the ultrafiltration method is the method of choice for efficient separation and accurate measurement of EE.

Exploiting or overcoming the dome trap for enhanced oral immunization and drug delivery.

The microfold cells (M cells) residing in the outmost follicle-associated epithelia (FAE) of Peyer's patches capture foreign particles and hand over to sub-FAE lymphatics, where the particles are retained and disposed subsequently. A concept of "dome trap" is proposed to highlight the significance of this mechanism. For oral immunization, it is better to exploit the entrapment capacity to maximize immune response, whereas for drug delivery it is better to overcome the dome trap to transport drugs into the systemic circulation. By optimizing the size, shape, surface charges and surface properties of particles, either oral immunization or drug delivery can be potentially enhanced.

Overcoming or circumventing the stratum corneum barrier for efficient transcutaneous immunization.

Transcutaneous immunization (TCI) is a promising alternative to vaccine delivery via the subcutaneous and intramuscular routes because of the unique immunological characteristics of the skin. However, the stratum corneum (SC) prevents entry of most therapeutic compounds into the body. Several physical devices have been developed to overcome the SC barrier, but still damage the skin. However, by targeting antigens to the abundant perifollicular antigen-presenting cells (APCs), the transfollicular route might be a promising approach for TCI without compromising the skin barrier.

Neuroprotective Effect of ß-Caryophyllene on Cerebral Ischemia-Reperfusion Injury via Regulation of Necroptotic Neuronal Death and Inflammation: In Vivo and in Vitro.

Necrotic cell death is a hallmark feature of ischemic stroke and it may facilitate inflammation by releasing intracellular components after cell-membrane rupture. Previous studies reported that ß-caryophyllene (BCP) mitigates cerebral ischemia-reperfusion (I/R) injury, but the underlying mechanism remains unclear. We explored whether BCP exerts a neuroprotective effect in cerebral I/R injury through inhibiting necroptotic cell death and inflammation. Primary neurons with and without BCP (0.2, 1, 5, 25 µM) treatment were exposed to oxygen-glucose deprivation and re-oxygenation (OGD/R). Neuron damage, neuronal death type and mixed lineage kinase domain-like (MLKL) protein expression were assessed 48 h after OGD/R. Furthermore, mice underwent I/R procedures with or without BCP (8, 24, 72 mg/kg, ip.). Neurologic dysfunction, cerebral infarct volumes, cell death, cytokine levels, necroptosis core molecules, and HMGB1-TLR4 signaling were determined at 48 h after I/R. BCP (5 µM) significantly reduced necroptotic neurons and MLKL protein expression following OGD/R. BCP (24, 72 mg/kg, ip.) reduced infarct volumes, neuronal necrosis, receptor-interaction protein kinase-1 (RIPK1), receptor-interaction protein kinase-3 (RIPK3) expression, and MLKL phosphorylation after I/R injury. BCP also decreased high-mobility group box 1 (HMGB1), toll-like receptor 4 (TLR4), interleukin-1ß (IL-1ß), and tumor necrosis factor-α (TNF-α) levels. Thus, BCP alleviates ischemic brain damage potentially by inhibiting necroptotic neuronal death and inflammatory response. This study suggests a novel application for BCP as a neuroprotective agent.

Ultrasound-Triggered Destruction of Folate-Functionalized Mesoporous Silica Nanoparticle-Loaded Microbubble for Targeted Tumor Therapy.

A multifunctional drug delivery vehicle, which combines the active targeted mesoporous silica nanoparticle (MSN) and microbubble (MB) drug delivery system, is proposed and fabricated. The resulting delivery vehicle integrates the merits of high drug loading capacity, multitargeting, and ultrasound-guided releasing. Folate (FA), which serves as an active ligand, is modified to the surface of MSN (MSN-FA) to enhance cell membrane translocation. MSN-FA is loaded with tanshinone IIA (TAN), then encapsulated in a microbubble (denoted as MSN-FA-TAN-MB) for more precise tumor targeting. The conjunction between FA and MSN is confirmed by fourier transform infrared spectroscopy (FTIR). The characteristics and morphology of MSN-FA-TAN-MB are investigated by confocal microscopy and transmission electron microscopy. In vitro cytotoxicity and cellular uptake studies of MSN-FA-TAN-MB are conducted on A549 and HeLa tumor cells. FA-facilitated MSN-FA-TAN uptake is shown by HeLa cells that overexpress FA receptors via a FA-receptor-mediated endocytosis mechanism. The ultrasound response property of MSN-FA-TAN-MB is also verified. MSN-FA-TAN-MB shows significant antitumor efficacy in vivo with the assistance of FA, MB, and an external ultrasound irradiation. Thus, this multifunctional vehicle may provide a novel strategy for tumor targeting and imaging in tumor therapy.

Glycyrrhetinic acid-functionalized mesoporous silica nanoparticles as hepatocellular carcinoma-targeted drug carrier.

In this study, a glycyrrhetinic acid-functionalized mesoporous silica nanoparticle (MSN-GA) was prepared for active tumor targeting. MSN-GA exhibited satisfactory loading capacity for insoluble drugs, uniform size distribution, and specific tumor cell targeting. Glycyrrhetinic acid, a hepatocellular carcinoma-targeting group, was covalently decorated on the surface of MSN via an amido bond. The successful synthesis of MSN-GA was validated by the results of Fourier transform infrared spectroscopy, transmission electron microscopy (TEM), and zeta potential measurement. TEM images revealed the spherical morphology and uniform size distribution of the naked MSN and MSN-GA. Curcumin (CUR), an insoluble model drug, was loaded into MSN-GA (denoted as MSN-GA-CUR) with a high-loading capacity (8.78%±1.24%). The results of the in vitro cellular experiment demonstrated that MSN-GA-CUR significantly enhanced cytotoxicity and cellular uptake toward hepatocellular carcinoma (HepG2) cells via a specific GA receptor-mediated endocytosis mechanism. The results of this study provide a promising nanoplatform for the targeting of hepatocellular carcinoma.

Novel multifunctional pH-sensitive nanoparticles loaded into microbubbles as drug delivery vehicles for enhanced tumor targeting.

This study fabricated novel multifunctional pH-sensitive nanoparticles loaded into microbubbles (PNP-MB) with the combined advantages of two excellent drug delivery vehicles, namely, pH-sensitive nanoparticles and microbubbles. As an antitumor drug, resveratrol (RES) was loaded into acetylated ß-cyclodextrin nanoparticles (RES-PNP). The drug-loaded nanoparticles were then encapsulated into the internal space of the microbubbles. The characterization and morphology of this vehicle were investigated through dynamic light scattering and confocal laser scanning microscopy, respectively. In vitro drug release was performed to investigate the pH sensitivity of RES-PNP. The antitumor property of RES-loaded PNP-MB (RES-PNP-MB) was also analyzed in vivo to evaluate the antitumor effect of RES-PNP-MB. Results suggested that PNP exhibited pH sensitivity, and was successfully encapsulated into the microbubbles. RES-PNP-MB exhibit effective tumor growth suppressing in vivo. Therefore, such drug delivery vehicle should be of great attention in tumor therapy.