Effects of Surface IR783 Density on the In Vivo Behavior and Imaging Performance of Liposomes.

Background: Nanoparticles conjugated with fluorescent probes have versatile applications, serving not only for targeted fluorescent imaging but also for evaluating the in vivo profiles of designed nanoparticles. However, the relationship between fluorophore density and nanoparticle behavior remains unexplored. Methods: The IR783-modified liposomes (IR783-sLip) were prepared through a modified ethanol injection and extrusion method. The cellular uptake efficiency of IR783-sLip was characterized by flow cytometry and fluorescence microscope imaging. The effects of IR783 density on liposomal in vivo behavior were investigated by pharmacokinetic studies, biodistribution studies, and in vivo imaging. The constitution of protein corona was analyzed by the Western blot assay. Results: Dense IR783 modification improved cellular uptake of liposomes in vitro but hindered their blood retention and tumor imaging performance in vivo. We found a correlation between IR783 density and protein corona absorption, particularly IgM, which significantly impacted the liposome performance. Meanwhile, we observed that increasing IR783 density did not consistently improve the effectiveness of tumor imaging. Conclusions: Increasing the density of modified IR783 on liposomes is not always beneficial for tumor near-infrared (NIR) imaging yield. It is not advisable to prematurely evaluate novel nanomaterials through fluorescence dye conjugation without carefully optimizing the density of the modifications.

Optimized RNA interference therapeutics combined with interleukin-2 mRNA for treating hepatitis B virus infection.

This study aimed to develop a pan-genotypic and multifunctional small interfering RNA (siRNA) against hepatitis B virus (HBV) with an efficient delivery system for treating chronic hepatitis B (CHB), and explore combined RNA interference (RNAi) and immune modulatory modalities for better viral control. Twenty synthetic siRNAs targeting consensus motifs distributed across the whole HBV genome were designed and evaluated. The lipid nanoparticle (LNP) formulation was optimized by adopting HO-PEG2000-DMG lipid and modifying the molar ratio of traditional polyethylene glycol (PEG) lipid in LNP prescriptions. The efficacy and safety of this formulation in delivering siHBV (tLNP/siHBV) along with the mouse IL-2 (mIL-2) mRNA (tLNP/siHBVIL2) were evaluated in the rAAV-HBV1.3 mouse model. A siRNA combination (terms "siHBV") with a genotypic coverage of 98.55% was selected, chemically modified, and encapsulated within an optimized LNP (tLNP) of high efficacy and security to fabricate a therapeutic formulation for CHB. The results revealed that tLNP/siHBV significantly reduced the expression of viral antigens and DNA (up to 3log10 reduction; vs PBS) in dose- and time-dependent manners at single-dose or multi-dose frequencies, with satisfactory safety profiles. Further studies showed that tLNP/siHBVIL2 enables additive antigenic and immune control of the virus, via introducing potent HBsAg clearance through RNAi and triggering strong HBV-specific CD4+ and CD8+ T cell responses by expressed mIL-2 protein. By adopting tLNP as nucleic acid nanocarriers, the co-delivery of siHBV and mIL-2 mRNA enables synergistic antigenic and immune control of HBV, thus offering a promising translational therapeutic strategy for treating CHB.

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.

Design, Synthesis, Formulation, and Bioevaluation of Trisubstituted Triazines as Highly Selective mTOR Inhibitors for the Treatment of Human Breast Cancer.

The aberrant activation of the PI3K/mTOR signaling pathway is implicated in various human cancers. Thus, the development of inhibitors targeting mTOR has attracted considerable attention. In this study, we used a structure-based drug design strategy to discover a highly potent and kinase-selective mTOR inhibitor 24 (PT-88), which demonstrated an mTOR inhibitory IC50 value of 1.2 nM without obvious inhibition against another 195 kinases from the kinase profiling screening. PT-88 displayed selective inhibition against MCF-7 cells (IC50: 0.74 µM) with high biosafety against normal cells, in which autophagy induced by mTOR inhibition was implicated. After successful encapsulation in a lipodisc formulation, PT-88 demonstrated favorable pharmacokinetic and biosafety profiles and exerted a large antitumor effect in an MCF-7 subcutaneous bearing nude mice model. Our study shows the discovery of a highly selective mTOR inhibitor using a structure-based drug discovery strategy and provides a promising antitumor candidate for future study and development.

mIgM-mediated splenic marginal zone B cells targeting of folic acid for immunological evasion.

Folic acid is a fully oxidized synthetic folate with high bioavailability and stability which has been extensively prescribed to prevent congenital disabilities. Here we revealed the immunosuppressive effect of folic acid by targeting splenic marginal zone B (MZB) cells. Folic acid demonstrates avid binding with the Fc domain of immunoglobulin M (IgM), targeting IgM positive MZB cells in vivo to destabilize IgM-B cell receptor (BCR) complex and block immune responses. The induced anergy of MZB cells by folic acid provides an immunological escaping window for antigens. Covalent conjugation of folic acid with therapeutic proteins and antibodies induces immunological evasion to mitigate the production of anti-drug antibodies, which is a major obstacle to the long-term treatment of biologics by reducing curative effects and/or causing adverse reactions. Folic acid acts as a safe and effective immunosuppressant via IgM-mediated MZB cells targeting to boost the clinical outcomes of biologics by inhibiting the production of anti-drug antibodies, and also holds the potential to treat other indications that adverse immune responses need to be transiently shut off.

A natural IgM hitchhiking strategy for delivery of cancer nanovaccines to splenic marginal zone B cells.

B cell-targeted cancer vaccines are receiving increasing attention in immunotherapy due to the combined antibody-secreting and antigen-presenting functions. In this study, we propose a natural IgM-hitchhiking delivery strategy to co-deliver tumor antigens and adjuvants to splenic marginal zone B (MZB) cells. We constructed nanovaccines (FA-sLip/OVA/MPLA) consisting of classical folic acid (FA)-conjugated liposomes co-loaded with ovalbumin (OVA) and toll-like receptor 4 agonists, MPLA. We found that natural IgM absorption could be manipulated at the bio-nano interface on FA-sLip/OVA/MPLA, enabling targeted delivery to splenic MZB cells. Systemic administration of FA-sLip/OVA/MPLA effectively activated splenic MZB cells via IgM-mediated multiplex pathways, eliciting antigen-specific humoral and cytotoxic T lymphocyte responses, and ultimately retarding E.G7-OVA tumor growth. In addition, combining FA-sLip/OVA/MPLA immunization with anti-PD-1 treatments showed improved antitumor efficiency. Overall, this natural IgM-hitchhiking delivery strategy holds great promise for efficient, splenic MZB cell-targeted delivery of cancer vaccines in future applications.

Effects of PEG antibodies on in vivo performance of LNP-mRNA vaccines.

Polyethylene glycol (PEG) plays important roles in stabilizing and lengthening circulation time of lipid nanoparticle (LNP) vaccines. Nowadays various levels of PEG antibodies have been detected in human blood, but the impact and mechanism of PEG antibodies on the in vivo performance of LNP vaccines has not been clarified thoroughly. By illustrating the distribution characteristics of PEG antibodies in human, the present study focused on the influence of PEG antibodies on the safety and efficacy of LNP-mRNA vaccine against COVID-19 in animal models. It was found that PEG antibodies led to shortened blood circulation duration, elevated accumulation and mRNA expression in liver and spleen, enhanced expression in macrophage and dendritic cells, while without affecting the production of anti-Spike protein antibodies of COVID-19 LNP vaccine. Noteworthily, PEG antibodies binding on the LNP vaccine increased probability of complement activation in animal as well as in human serum and led to lethal side effect in large dosage via intravenous injection of mice. Our data suggested that PEG antibodies in human was a risky factor of LNP-based vaccines for biosafety concerns but not efficacy.

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.

Protein corona on brain targeted nanocarriers: Challenges and prospects.

Safe and efficient medical therapy for brain diseases is still an unmet clinical need due to various barriers represented by the blood-brain barrier. Well-designed brain targeted nanocarriers are potential solutions for enhanced brain drug delivery; however, the complicated in vivo process attenuates performance of nanocarriers, which severely hampers clinical translation. The formation of protein corona (PC) is inevitable for nanocarriers circulation and transport in biofluids, acting as an important factor to regulate in vivo performance of nanocarriers. In this review, the reported strategies have been retrospected for better understanding current situation in developing brain targeted nanocarriers. The interplay between brain targeted nanocarriers and plasma proteins is emphasized to comprehend how the nanocarriers adsorb proteins by certain synthetic identity, and following regulations on in vivo performance of nanocarriers. More importantly, the mainstream methods to promote efficiency of nanocarriers by regulating PC, defined as in vitro functionalization and in vivo functionalization strategies, are also discussed. Finally, viewpoints about future development of brain targeted nanocarriers according to the understanding on nanocarriers-PC interaction are proposed.

Interplay between Liposomes and IgM: Principles, Challenges, and Opportunities.

Liposomes have received tremendous attention as a class of versatile pharmaceutical vehicles of great potential over the past several decades. However, the application of liposomes encounters major challenges due to the knowledge gaps in their in vivo delivery process. Immunoglobulin M (IgM) displays both pervasiveness and complexity in regulating the biological functions as well as eliciting adverse effects of liposomes. Understanding, mitigating, and exploiting the duality of IgM are prerequisites for achieving various biomedical applications of liposomes. In this review, the intricate relationship between liposomes and their biological environments has been summarized, with an emphasis on the regulatory effects of IgM on in vivo performance of liposomes. Corresponding solutions have also been discussed to evade IgM-mediated opsonization for safe and efficient drug delivery.

Albumin-Hitchhiking Drug Delivery to Tumor-Draining Lymph Nodes Precisely Boosts Tumor-Specific Immunity through Autophagy Modulation of Immune Cells.

Tumor-draining lymph nodes (TDLNs) are the first sites where tumor components reach and dendritic cells (DCs) present tumor-associated antigens to T cells. DCs rely on autophagy to process tumor antigens into epitope peptides to form epitope-MHC complexes. Selective delivery of autophagy-stimulating drugs to TDLNs may be a precise strategy to boost chemotherapy-induced antitumor immunity. Here, a multistage stimulating strategy is proposed to activate the antitumor immunity cascade by inducing immunogenic death of tumor cells and elevating antigen presentation of DCs in TDLNs. A tumor-microenvironment-responsive "albumin-hitchhiking" micelle is established by self-assembling tumor-targeting oxaliplatin prodrug and lipophilized trehalose prodrug. This demonstrates that lipophilic modification of trehalose with a DSPE tail and the precise exposure in the tumor site enhances its binding to endogenous albumin and realizes TDLNs-selective reflux, where it upregulates antigen processing and presentation of DCs. This study introduces an approach for targeted delivery to TDLNs and provides insights into mechanisms of autophagy in tumor-specific immunity.

Lipid-Based Nanocarriers Enabled Oral Delivery of Oleanolic Acid Derivative DKS26 for Diabetes Management.

Oleanolic acid derivative DKS26 has hypolipidemic, islet, and hepatoprotective effects. However, high lipophilicity and low water solubility led to DKS26 extremely low oral bioavailability. Herein, lipid-based nanocarriers, including lipid nanodiscs (sND/DKS26) and liposomes (sLip/DKS26), are prepared to improve DKS26 oral absorption. In comparison to free DKS26 (5.81%), the absolute oral bioavailabilities are significantly increased to 29.47% (sND/DKS26) and 37.25% (sLip/DKS26) without detectable toxicity or immunogenicity even after repeated administrations. Both sND/DKS26 and sLip/DKS26 significantly reduce the feeding glucose level and the AUC of OGTT in db/db diabetic mice. Aiding by the newly developed scFv-based nanocarrier separation methods, no intact nanocarriers are detected in blood circulation after oral administration, suggesting that both formulations are unable to penetrate the intestinal epithelium. They enhance DKS26 absorption mainly by improving intestinal cell uptake and rapid intracellular release of the payload. Since pre-existing anti-PEG is widely detected in humans, the present oral absorption pathway of both nanocarriers successfully avoids unfavorable immunological responses after interaction with anti-PEG antibodies. The application of lipid-based nanocarriers paves an efficient and safe avenue for the clinical translation and application of poorly soluble therapeutics derived from traditional Chinese medicine.

Photoresponsive prodrug-dye nanoassembly for in-situ monitorable cancer therapy.

Photocleavable prodrugs enable controllable drug delivery to target sites modulated by light irradiation. However, the in vivo utility is usually hindered by their insolubility and inefficient delivery. In this study, we report a simple strategy of co-assembling boron-dipyrromethene-chlorambucil prodrug and near-infrared dye IR783 to fabricate photoresponsive nanoassemblies, which achieved both high prodrug loading capacity (~99%) and efficient light-triggered prodrug activation. The incorporated IR783 dye not only stabilized the nanoparticles and contributed tumor targeting as usual, but also exhibited degradation after light irradiation and in-situ monitoring of nanoparticle dissociation by fluorescent imaging. Systemic administration of the nanoparticles and localized light irradiation at tumor sites enabled monitorable and efficient drug release in vivo. Our results demonstrate that such prodrug-dye co-assembled nanomedicine is a promising formulation for photoresponsive drug delivery, which would advance the translation of photoresponsive nanomedicines.

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.

Engineered platelets-based drug delivery platform for targeted thrombolysis.

Thrombolytic agents have thus far yielded limited therapeutic benefits in the treatment of thrombotic disease due to their short half-life, low targeting ability, and association with serious adverse reactions, such as bleeding complications. Inspired by the natural roles of platelets during thrombus formation, we fabricated a platelet-based delivery system (NO@uPA/PLTs) comprising urokinase (uPA) and arginine (Arg) for targeted thrombolysis and inhibition of re-embolism. The anchoring of uPA to the platelet surface by lipid insertion increased the thrombotic targeting and in vivo circulation duration of uPA without disturbing platelet functions. Nitric oxide (NO) generated by the loaded Arg inhibited platelet aggregation and activation at the damaged blood vessel, thereby inhibiting re-embolism. NO@uPA/PLTs effectively accumulated at the thrombi in pulmonary embolism and carotid artery thrombosis model mice and exerted superior thrombolytic efficacy. In addition, the platelet delivery system showed excellent thrombus recurrence prevention ability in a mouse model of secondary carotid artery injury. The coagulation indicators in vivo showed that the platelet-based uPA and NO co-delivery system possessed a low hemorrhagic risk, providing a promising tool for rapid thrombolysis and efficient inhibition of posttreatment re-embolism.

Peptide-decorated nanocarriers penetrating the blood-brain barrier for imaging and therapy of brain diseases.

Blood-Brain Barrier (BBB) is one of the most important physiological barriers strictly restricting the substance exchange between blood and brain tissues. While the BBB protects the brain from infections and toxins and maintains brain homeostasis, it is also recognized as the main obstacle to the penetration of therapeutics and imaging agents into the brain. Due to high specificity and affinity, peptides are frequently exploited to decorate nanocarriers across the BBB for diagnosis and/or therapy purposes. However, there are still some challenges that restrict their clinical application, such as stability, safety and immunocompatibility. In this review, we summarize the biological and pathophysiological characteristics of the BBB, strategies across the BBB, and recent progress on peptide decorated nanocarriers for brain diseases diagnosis and therapy. The challenges and opportunities for their translation are also discussed.

Protein corona: challenges and opportunities for targeted delivery of nanomedicines.

INTRODUCTION: Targeted drug delivery has been widely explored as a promising way to improve the performance of nanomedicines. However, protein corona formed on the nano-surface represents a major issue that has great impacts on the in vivo fate of targeting nanomedicines, which has been overlooked in the past. With the increasing understanding of protein corona in the recent decade, many efforts have been made to improve targeting efficacy. AREAS COVERED: In this review, we briefly summarize insights of targeted delivery systems inspired by protein corona, and discuss the promising strategies to regulate protein corona for better targeting. EXPERT OPINION: The interaction between nanomedicines and endogenous proteins brings great uncertainty and challenges, but it also provides great opportunities for the development of targeting nanomedicines at the same time. With increasing understanding of protein corona, the strategies to regulate protein corona pave new avenues for the development of targeting nanomedicines.

Evaluation of CTB-sLip for Targeting Lung Metastasis of Colorectal Cancer.

Lung metastasis of colorectal cancer is common in the clinic; however, precise targeting for the diagnosis and therapy purposes of those lung metastases remains challenging. Herein, cholera toxin subunit b (CTB) protein was chemically conjugated on the surface of PEGylated liposomes (CTB-sLip). Both human-derived colorectal cancer cell lines, HCT116 and HT-29, demonstrated high binding affinity and cellular uptake with CTB-sLip. In vivo, CTB-sLip exhibited elevated targeting capability to the lung metastasis of colorectal cancer in the model nude mice in comparison to PEGylated liposomes (sLip) without CTB modification. CTB conjugation induced ignorable effects on the interaction between liposomes and plasma proteins but significantly enhanced the uptake of liposomes by numerous blood cells and splenic cells, leading to relatively rapid blood clearance in BALB/c mice. Even though repeated injections of CTB-sLip induced the production of anti-CTB antibodies, our results suggested CTB-sLip as promising nanocarriers for the diagnosis of lung metastasis of colorectal cancer.

Topical instillation of cell-penetrating peptide-conjugated melphalan blocks metastases of retinoblastoma.

Retinoblastoma is the most common primary intraocular malignancy in infancy with a metastases-related death risk. However, a safe and convenient treatment without enucleation is still an unmet clinical need. In this work, a cell-penetrating peptide, 89WP, was conjugated with melphalan (89WP-Mel), which achieved high tumor inhibition effects as intravitreally injected melphalan via topical instillation for the first time. Notably, the "outside-in" diffusion of instilled 89WP-Mel created a protective shield surrounding the eye, efficiently preventing tumor metastases, while the mice treated with intravitreally injected melphalan suffered more brain metastases related death. The ocular absorption of 89WP-conjugated melphalan and other small molecules, both hydrophobic and hydrophilic, occurred via non-corneal pathway with high safety and a prolonged residence duration in retina up to 24 h. The present work paves a new avenue for simultaneous intraocular tumor inhibition and extraocular metastases prevention in a safe and convenient way via topical instillation.

cRGD enables rapid phagocytosis of liposomal vancomycin for intracellular bacterial clearance.

RGD motif has long been exploited as a versatile tool for targeted drug delivery. However, there are so far no successful clinical translations of RGD functionalized nanomedicines. The lack of comprehensive understanding of their in vivo delivery process poses one of the main obstacles. As a reflection on cRGD-enabled targeting delivery, herein the in vivo fate of cyclic RGD peptide functionalized liposome (cRGD-sLip) and its fundamental mechanism are investigated. cRGD-sLip demonstrates incredibly rapid blood clearance and massive mononuclear phagocytic system (MPS) accumulation after intravenous injection. Phagocytes actively capture cRGD-sLip by recognizing αvß3 integrins and scavenger receptors, urging reinterrogation of RGD enabled targeting delivery. Intracellular infection with microbes invading and persisting in the phagocytic system poses serious threats to global public health. Most antimicrobial agents are unable to penetrate through host cell membrane and achieve optimal intracellular therapeutic concentration, resulting in ineffective bacterial killing. By leveraging the rapid phagocytic uptake, cRGD-sLip demonstrates the capability to facilitate effective targeted drug delivery to bacteria infected macrophages and successfully reduce the bacterial burden in a murine intracellular Methicillin-resistant Staphylococcus aureus (MRSA) infection model, verifying the potential value of cRGD-sLip in improving therapeutic efficacy of existing antibiotics in the treatment of intracellular bacterial infection.