Phospholipid Type Regulates Protein Corona Composition and In Vivo Performance of Lipid Nanodiscs.

Over the years, there has been significant interest in PEGylated lipid-based nanocarriers within the drug delivery field. The inevitable interplay between the nanocarriers and plasma protein plays a pivotal role in their in vivo biological fate. Understanding the factors influencing lipid-based nanocarrier and protein corona interactions is of paramount importance in the design and clinical translation of these nanocarriers. Herein, discoid-shaped lipid nanodiscs (sNDs) composed of different phospholipids with varied lipid tails and head groups were fabricated. We investigated the impact of phospholipid components on the interaction between sNDs and serum proteins, particle stability, and biodistribution. The results showed that all of these lipid nanodiscs remained stable over a 15 day storage period, while their stability in the blood serum demonstrated significant differences. The sND composed of POPG exhibited the least stability due to its potent complement activation capability, resulting in rapid blood clearance. Furthermore, a negative correlation between the complement activation capability and serum stability was identified. Pharmacokinetic and biodistribution experiments indicated that phospholipid composition did not influence the capability of sNDs to evade the accelerated blood clearance phenomenon. Complement deposition on the sND was inversely associated with the area under the curve. Additionally, all lipid nanodiscs exhibited dominant adsorption of apolipoprotein. Remarkably, the POPC-based lipid nanodisc displayed a significantly higher deposition of apolipoprotein E, contributing to an obvious brain distribution, which provides a promising tool for brain-targeted drug delivery.

Reexamining the effects of drug loading on the in vivo performance of PEGylated liposomal doxorubicin.

Higher drug loading employed in nanoscale delivery platforms is a goal that researchers have long sought after. But such viewpoint remains controversial because the impacts that nanocarriers bring about on bodies have been seriously overlooked. In the present study we investigated the effects of drug loading on the in vivo performance of PEGylated liposomal doxorubicin (PLD). We prepared PLDs with two different drug loading rates: high drug loading rate, H-Dox, 12.9% w/w Dox/HSPC; low drug loading rate, L-Dox, 2.4% w/w Dox/HSPC (L-Dox had about 5 folds drug carriers of H-Dox at the same Dox dose). The pharmaceutical properties and biological effects of H-Dox and L-Dox were compared in mice, rats or 4T1 subcutaneous tumor-bearing mice. We showed that the lowering of doxorubicin loading did not cause substantial shifts to the pharmaceutical properties of PLDs such as in vitro and in vivo stability (stable), anti-tumor effect (equivalent effective), as well as tissue and cellular distribution. Moreover, it was even more beneficial for mitigating the undesired biological effects caused by PLDs, through prolonging blood circulation and alleviating cutaneous accumulation in the presence of pre-existing anti-PEG Abs due to less opsonins (e.g. IgM and C3) deposition on per particle. Our results warn that the effects of drug loading would be much more convoluted than expected due to the complex intermediation between nanocarriers and bodies, urging independent investigation for each individual delivery platform to facilitate clinical translation and application.

Folic Acid Enables Targeting Delivery of Lipodiscs by Circumventing IgM-Mediated Opsonization.

Folic acid (FA) is one of the most widely utilized small-molecule ligands for cancer targeted drug delivery. Natural IgM was recently found to avidly absorb on the surface of FA-functionalized liposomes (FA-sLip), negatively regulating the in vivo performance by efficiently activating complement. Herein, FA-functionalized lipodiscs (FA-Disc) were constructed to successfully circumvent IgM-mediated opsonization and retained binding activity with folate receptors in vivo. The FA moiety along with the bound IgM was restricted to the highly curved rim of lipodiscs, leading to IgM incapability of presenting the membrane-bound conformation to trigger complement activation. The C1q docking, C3 binding, and C5a release were blocked and accelerated blood clearance phenomenon was mitigated of FA-Disc. FA-Disc retained folate binding activity and could effectively target folate receptor positive tumors in vivo. The present study provides a useful solution to avoid the negative regulation by IgM and achieve FA-enabled targeting by exploring disc-shaped nanocarriers.

A Nanoantidote Alleviates Glioblastoma Chemotoxicity without Efficacy Compromise.

Cancer patients suffer from the toxicity of chemotherapy. Antidote, given as a remedy limiting poison, is an effective way to counteract toxicity. However, few antidotes abrogate chemotoxicity without compromising the therapeutic efficacy. Herein, a rationally designed nanoantidote can neutralize chemo-agents in normal cells but not enter tumors and thus would not interfere with the efficacy of tumor treatment. The nanoantidote, consisting of a dendrimer core wrapped by reductive cysteine, captures Temozolomide (TMZ, the glioblastoma standard chemotherapy). Meanwhile, thanks to the blood-brain barrier (BBB) and the size of the nanoantidote, the nanoantidote cannot enter glioblastoma. In murine models, the nanoantidote distributes in normal tissues without crossing the BBB, so it markedly reduces the chemotoxicity of TMZ and retains the original TMZ therapeutic efficacy. With most nanotechnologies focusing on antitumor treatment, this detoxicating strategy demonstrates a nanoplatform to reduce chemotoxicity using physiology barriers and introduces a new approach to nanomedicine for cancer chemotherapy.

Facile Separation of PEGylated Liposomes Enabled by Anti-PEG scFv.

PEGylated nanocarriers have gained increasing attention due to reduced toxicity and enhanced circulation compared with free drugs. According to guidances of drug regulatory departments worldwide, it is crucial to determine free and liposomal drug concentrations; however, the conventional used separation methods including dialysis, ultrafiltration, and solid-phase extraction (SPE) have drawbacks of time-consuming, drug leakage, environmental pollution or error bias of trace level drug. Here we developed a facile PEG-scFv-based separation method combined with HPLC to quantify free doxorubicin (DOX) and liposomal DOX in plasma. Anti-PEG single chain variable fragment antibody (PEG-scFv) was adopted to sediment PEGylated liposomes by simple incubation and low speed centrifugation. Compared to SPE, it demonstrated sufficient accuracy and sensitivity to evaluate free and liposomal DOX with intact liposomes. Therefore, it can serve as an alternative approach of SPE, which is suitable for quality assessment and pharmacokinetics evaluation of PEGylated liposomal drugs and possible other PEGylated nanocarriers.

Self-Adjuvant Effect by Manipulating the Bionano Interface of Liposome-Based Nanovaccines.

Nanovaccines are of increasing scrutiny due to their plasticity in size, composition, and surface properties to enhance antigenicity. However, inevitable absorption of plasma proteins affects the in vivo fate of nanovaccines by reshaping biological identity. Herein IgM was validated as a self-adjuvant by regulating antigen-presenting cells recognition of liposome-based nanovaccines. DCDX-modified liposomes with loading of ovalbumin (DCDX-sLip/OVA) heavily absorbed IgM via electrostatic interaction, demonstrating significant splenic B cells targeting. IgM absorbed on DCDX-sLip/OVA enhanced antigen uptake and presentation by both IgM-complement and IgM-FcµR pathways. DCDX-sLip/OVA induced a stronger IgG1 titer than ovalbumin-loaded plain liposomes (sLip/OVA) while maintaining a comparably high level of IgG2a titer with high biosafety, indicating that IgM absorption after DCDX modification could improve the antigenicity by enhancing the Th2-polarized immune response. The present work suggested manipulation of IgM absorption may provide a new impetus to improve in vivo performance of nanovaccines.

Deciphering Protein Corona by scFv-Based Affinity Chromatography.

It remains challenging to precisely decipher the structural and functional characteristics of protein coronas. To overcome the drawbacks frequently occurring in the traditional separation methods, an anti-PEG single-chain variable fragment (PEG-scFv) based affinity chromatography (AfC) was developed to achieve precise and efficient separation of protein coronas on PEGylated liposomes (sLip). His-tagged PEG-scFv could readily capture sLip without affecting protein corona compositions, and separate sLip/protein complex from plasma protein aggregates and endogenous vesicles through the Ni-NTA column. AfC demonstrated 43-fold higher protein corona collecting efficiency than centrifugation, which was extremely crucial for separation of in vivo protein coronas due to the limitation of sample size. AfC evaded contamination by endogenous vesicles and protein aggregates occurring in centrifugation, and reserved the loosely bound proteins, providing an unprecedented approach to deeply decipher protein coronas. The scFv-based AfC also paves new avenues for the separation of protein coronas formed on other nanomedicines.

Factors Influencing the Immunogenicity and Immunotoxicity of Cyclic RGD Peptide-Modified Nanodrug Delivery Systems.

c(RGDyK)-modified liposomes have been shown to be immunogenic and potentially trigger acute systemic anaphylaxis upon repeated intravenous injection in both BALB/c nude mice and ICR mice. However, questions concerning the potential influence of mouse strains, immunization routes, drug carrier properties, and changes in c(RGDyK) itself on the immunogenicity and resultant immunotoxicity (anaphylaxis) of cyclic RGD peptide-modified nanodrug delivery systems remain unanswered. Here, these potential impact factors were investigated, aiming to better understand the immunological properties of cyclic RGD peptide-based nanodrug delivery systems and seek for solutions for this immunogenicity-associated issue. It was revealed that anaphylaxis caused by intravenous c(RGDyK)-modified drug delivery systems might be avoided by altering the preimmunization route (i.e., subcutaneous injection), introducing positively charged lipids into the liposomes and by using micelles or red blood cell membrane (RBC)-based drug delivery systems as the carrier. Different murine models showed different incidences of anaphylaxis following intravenous c(RGDyK)-liposome stimulation: anaphylaxis was not observed in both SD rats and BALB/c mice and was less frequent in C57BL/6 mice than that in ICR mice. In addition, enlarging the peptide ring of c(RGDyK) by introducing amino sequence serine-glycine-serine reduced the incidence of anaphylaxis post the repeated intravenous c(RGDyKSGS)-liposome stimulation. However, immunogenicity of cyclic RGD-modified drug carriers could not be reversed, although some reduction in IgG antibody production was observed when ICR mice were intravenously stimulated with c(RGDyK)-modified micelles, RBC membrane-based drug delivery systems and c(RGDyKSGS)-liposomes instead of c(RGDyK)-liposomes. This study provides a valuable reference for future application of cyclic RGD peptide-modified drug delivery systems.

Octopus-like Flexible Vector for Noninvasive Intraocular Delivery of Short Interfering Nucleic Acids.

Gene therapy is promising for chronic posterior ocular diseases, which are causal factors for severe vision impairment and even blindness worldwide. However, the inherent absorption barriers of the eye restrict intraocular delivery of therapeutic nucleic acids via topical instillation. Safe and efficient nonviral vectors for ocular gene therapy are still unmet clinical desires. Herein, an octopus-like flexible multivalent penetratin (MVP) was designed to facilitate condensation and delivery of therapeutic nucleic acids using multiarm polyethylene glycol (PEG) as a core and conjugating penetratin at each end of the PEG arms as outspread tentacles. Among the MVPs, 8-valent penetratin (8VP) stably compacted nucleic acids into positively charged polyplexes smaller than 100 nm, promoting cellular uptake efficiency (approaching 100%) and transfection rate (over 75%). After being instilled into the conjunctival sac, 8VP enabled rapid (<10 min) and prolonged (>6 h) distribution of nucleic acids in the retina via a noncorneal pathway. In a retinoblastoma-bearing mice model, topical instillation of 8VP/siRNA efficiently inhibited the protein expression of intraocular tumor without toxicity. MVP is advantageous over the commercial transfection reagent in safety and efficiency, and therefore provides a promising vector for noninvasive intraocular gene delivery.

Oral Delivery of Honokiol Microparticles for Nonrapid Eye Movement Sleep.

Honokiol (HNK) is a small-molecule lignin extracted from Magnolia Officinalis, demonstrating high potency in promoting nonrapid eye movement (NREM) sleep by modulating the benzodiazepine site of the GABAA receptor. However, the clinical use of HNK in the treatment of insomnia is restricted by its extremely low oral bioavailability. In the present work, enhanced oral bioavailability of HNK was achieved by loading it into poly lactide-glycolide acid microparticles (HNK-MP). After oral administration, HNK-MP demonstrated 15-fold increase of AUC0-12 h in comparison to free HNK. The maximum blood concentration ( Cmax) of HNK in HNK-MP-treated rats was 3.6 µg/mL at 2 h after oral administration, which was 6.5-fold of that in free HNK-treated rats. Oral administration of HNK-MP (20 mg/kg) efficiently increased NREM sleep by 60% by enhancing the transition from wakefulness to NREM sleep in rats. The biosafety of HNK-MP was assessed in vivo, and no damage occurred in the gastrointestinal tract. The present study provides a promising oral HNK formulation for the treatment of insomnia.

Short Peptide-Mediated Brain-Targeted Drug Delivery with Enhanced Immunocompatibility.

Peptide ligands have been exploited as versatile tools to facilitate targeted delivery of nanocarriers. However, the effects of peptide ligands on immunocompatibility and therapeutic efficacy of liposomes remain intricate. Here, a short and stable brain targeted peptide ligand D8 was modified on the surface of doxorubicin-loaded liposomes (D8-sLip/DOX), demonstrating prolonged blood circulation and lower liver distribution in comparison to the long and stable D-peptide ligand DCDX-modified doxorubicin-loaded liposomes (DCDX-sLip/DOX) by mitigating natural IgM absorption. Despite the improved pharmacokinetic profiles, D8-sLip/DOX exhibited comparable brain targeting capacity in ICR mice and antiglioblastoma efficacy to DCDX-sLip/DOX in nude mice bearing intracranial glioblastoma. However, dramatic accumulation of DCDX-sLip/DOX in liver (especially during the first 8 h after intravenous injection) resulted in pathological symptoms, including nuclei swelling, necrosis of liver cells, and inflammation. These results suggest that short peptide ligand-mediated brain-targeted drug delivery systems possessing enhanced immunocompatibility are promising to facilitate efficient brain transport with improved biosafety.

A d-Peptide Ligand of Integrins for Simultaneously Targeting Angiogenic Blood Vasculature and Glioma Cells.

The current prognosis of glioma patients remains poor after intensive multimodal treatments, which is partially due to the existence of the blood-brain tumor barrier (BBTB). In the present study, a novel "bifunctional ligand" (termed DVS) was developed by retro-inverso isomerization. DVS is a ligand of integrins highly expressed on glioma cells and tumor neovasculature. DVS exhibited exceptional stability in serum and demonstrated significantly higher targeting efficiency for glioma and HUVEC cells compared with the parent L-peptide. As a result, DVS modified micelles (DVS-MS) exhibited high encapsulation efficiency of doxorubicin, ideal size distribution, and sustained release behavior of the payload. In vivo studies showed that DVS-MS could target and efficiently deliver fluorescence to tumor cells and tumor vasculature not only in the mice bearing subcutaneous tumors but also in those bearing intracranial tumors. Moreover, doxorubicin loaded DVS modified micelles exerted potent tumor growth inhibitory activity against subcutaneous and intracranial human glioma in comparison to drug loaded plain micelles and LVS modified micelles. Therefore, DVS appears to be a suitable targeting ligand with potential applications for glioma targeted drug delivery.

Enhanced Glioblastoma Targeting Ability of Carfilzomib Enabled by a DA7R-Modified Lipid Nanodisk.

The robust proliferation of tumors relies on a rich neovasculature for nutrient supplies. Therefore, a basic strategy of tumor targeting therapy should include not only killing regular cancer cells but also blocking tumor neovasculature. D-peptide DA7R, which was previously reported to specifically bind vascular endothelial growth factor receptor 2 (VEGFR2) and neuropilin-1 (NRP-1), could achieve the goal of multitarget recognition. Accordingly, the main purposes of this work were to establish a carfilzomib-loaded lipid nanodisk modified with multifunctional peptide DA7R (DA7R-ND/CFZ) and to evaluate its anti-glioblastoma efficacy in vitro and in vivo. It is testified that the DA7R peptide-conjugated lipid nanodisk can be specifically taken up by U87MG cells and HUVECs. Furthermore, DA7R-ND demonstrated a more enhanced penetration than that of the nonmodified formulation on the tumor spheroid model in vitro and more tumor region accumulation in vivo on the subcutaneous and intracranial tumor-bearing nude mice model. DA7R-ND was shown to co-localize with tumor neovasculature in vivo. When loaded with proteasome inhibitor carfilzomib, the DA7R-decorated nanodisk could remarkably suppress tumor proliferation, extend survival time of nude mice bearing an intracranial tumor, and inhibit neovasculature formation with an efficacy higher than that of the nonmodified nanodisk in vitro and in vivo. The present study verified that the heptapeptide DA7R-conjugated nanodisk is a promising nanocarrier for glioblastoma targeting therapy.

Repeatable and adjustable on-demand sciatic nerve block with phototriggerable liposomes.

Pain management would be greatly enhanced by a formulation that would provide local anesthesia at the time desired by patients and with the desired intensity and duration. To this end, we have developed near-infrared (NIR) light-triggered liposomes to provide on-demand adjustable local anesthesia. The liposomes contained tetrodotoxin (TTX), which has ultrapotent local anesthetic properties. They were made photo-labile by encapsulation of a NIR-triggerable photosensitizer; irradiation at 730 nm led to peroxidation of liposomal lipids, allowing drug release. In vitro, 5.6% of TTX was released upon NIR irradiation, which could be repeated a second time. The formulations were not cytotoxic in cell culture. In vivo, injection of liposomes containing TTX and the photosensitizer caused an initial nerve block lasting 13.5 ± 3.1 h. Additional periods of nerve block could be induced by irradiation at 730 nm. The timing, intensity, and duration of nerve blockade could be controlled by adjusting the timing, irradiance, and duration of irradiation. Tissue reaction to this formulation and the associated irradiation was benign.

Enhanced Triggering of Local Anesthetic Particles by Photosensitization and Photothermal Effect Using a Common Wavelength.

On-demand pain relief systems would be very helpful additions to the armamentarium of pain management. Near-infrared triggered drug delivery systems have demonstrated the potential to provide such care. However, challenges remain in making such systems as stimulus-sensitive as possible, to enhance depth of tissue penetration, repeatability of triggering, and safety. Here we developed liposomes containing the local anesthetic tetrodotoxin and also containing a photosensitizer and gold nanorods that were excitable at the same near-infrared wavelength. The combination of triggering mechanisms enhanced the photosensitivity and repeatability of the system in vitro when compared with liposomes with a single photoresponsive component. In vivo, on-demand local anesthesia could be induced with a low irradiance and short irradiation duration, and liposomes containing both photosensitizer and gold nanorods were more effective than those containing just one photoresponsive component. Tissue reaction was benign.

Ultrasensitive Phototriggered Local Anesthesia.

An injectable local anesthetic producing repeatable on-demand nerve block would be desirable for pain management. Here we present a phototriggerable device to achieve repeatable and adjustable on-demand local anesthesia in superficial or deep tissues, consisting of gold nanorods attached to low temperature sensitive liposomes (LTSL). The particles were loaded with tetrodotoxin and dexmedetomidine. Near-infrared light (NIR, 808 nm, continuous wave) could heat gold nanorods at low fluence (short duration and low irradiance), leading to rapid release of payload. In vivo, 1-2 min of irradiation at ≤272 mW/cm2 produced repeatable and adjustable on-demand infiltration anesthesia or sciatic nerve blockade with minimal toxicity. The nerve block intensity and duration correlated with the irradiance and duration of the applied light.

Phototriggered Drug Delivery Using Inorganic Nanomaterials.

Light has many desirable properties as the stimulus for triggerable drug delivery systems. Inorganic nanomaterials are often key components in transducing light into drug delivery events. The nature of the light and the inorganic materials can affect the efficacy and safety of the drug delivery system.

Glioma-Targeted Drug Delivery Enabled by a Multifunctional Peptide.

The rapid proliferation of glioma relies on vigorous angiogenesis for the supply of essential nutrients; thus, a radical method of antiglioma therapy should include blocking tumor neovasculature formation. A phage display selected heptapeptide, the glioma-initiating cell peptide GICP, was previously reported as a ligand of VAV3 protein (a Rho GTPase guanine nucleotide exchange factor), which is overexpressed on glioma cells and tumor neovasculature. Therefore, GICP holds potential for the multifunctional targeting of glioma (tumor cells and neovasculature). We developed GICP-modified micelle-based paclitaxel delivery systems for antiglioma therapy in vitro and in vivo. GICP and GICP-modified PEG-PLA micelles (GICP-PEG-PLA) could be significantly taken up by U87MG cells, a human cell line derived from malignant gliomas and human umbilical vein endothelial cells (HUVECs). Furthermore, GICP-PEG-PLA micelles demonstrated enhanced penetration in a tumor spheroid model in vitro in comparison to unmodified micelles. In vivo, DiR-loaded GICP-PEG-PLA micelles exhibited superior accumulation in the tumor region by targeting neovasculature and glioma cells in nude mice bearing subcutaneous glioma. When loaded with paclitaxel, GICP-PEG-PLA micelles could more effectively suppress tumor growth and neovasculature formation than unmodified micelles in vivo. Our results indicated that GICP could serve as a promising multifunctional ligand for glioma targeting.

Tetrodotoxin, Epinephrine, and Chemical Permeation Enhancer Combinations in Peripheral Nerve Blockade.

BACKGROUND: Chemical permeation enhancers (CPEs) have the potential to improve nerve blockade by site 1 sodium channel blockers such as tetrodotoxin (TTX). Here, we investigated the efficacy and toxicity of CPE-enhanced nerve blockade across a range of TTX concentrations using 2 CPEs (sodium octyl sulfate and octyl trimethyl ammonium bromide). We also tested the hypothesis that CPEs could be used to reduce the concentrations of TTX and/or of a second adjuvant drug (in this case, epinephrine) needed to achieve prolonged local anesthesia METHODS:: Sprague-Dawley rats were injected at the sciatic nerve with combinations of TTX and CPEs, with and without epinephrine. Sensory and motor nerve blockade were assessed using a modified hot plate test and a weight-bearing test, respectively. Systemic and local toxicities of the different combinations were assessed. RESULTS: Addition of increasing concentrations of TTX to fixed concentrations of CPEs produced a marked concentration-dependent improvement in the rate of successful nerve blocks and in nerve block duration. CPEs did not affect systemic toxicity. At some concentrations, the addition of sodium octyl sulfate increased the duration of block from TTX plus epinephrine, and epinephrine increased that from TTX plus CPEs. The addition of epinephrine did not cause an increase in local toxicity, and it markedly reduced systemic toxicity. CONCLUSIONS: CPEs can prolong the duration of nerve blockade across a range of concentrations of TTX. CPEs could also be used to reduce the concentration of epinephrine needed to achieve a given degree of nerve block. CPEs may be useful in enhancing nerve blockade from site 1 sodium channel blockers.

Enhanced Precision of Nanoparticle Phototargeting in Vivo at a Safe Irradiance.

A large proportion of the payload delivered by nanoparticulate therapies is deposited not in the desired target destination but in off-target locations such as the liver and spleen. Here, we demonstrate that phototargeting can improve the specific targeting of nanoparticles to tumors. The combination of efficient triplet-triplet annihilation upconversion (TTA-UC) and Förster resonance energy transfer (FRET) processes allowed in vivo phototargeting at a safe irradiance (200 mW/cm(2)) over a short period (5 min) using green light.