Prevention of prostate cancer metastasis by a CRISPR-delivering nanoplatform for interleukin-30 genome editing.

Prostate cancer (PC) is a leading cause of cancer-related deaths in men worldwide. Interleukin-30 (IL-30) is a PC progression driver, and its suppression would be strategic for fighting metastatic disease. Biocompatible lipid nanoparticles (NPs) were loaded with CRISPR-Cas9gRNA to delete the human IL30 (hIL30) gene and functionalized with anti-PSCA-Abs (Cas9hIL30-PSCA NPs). Efficiency of the NPs in targeting IL-30 and the metastatic potential of PC cells was examined in vivo in xenograft models of lung metastasis, and in vitro by using two organ-on-chip (2-OC)-containing 3D spheroids of IL30+ PC-endothelial cell co-cultures in circuit with either lung-mimicking spheroids or bone marrow (BM)-niche-mimicking scaffolds. Cas9hIL30-PSCA NPs demonstrated circulation stability, genome editing efficiency, without off-target effects and organ toxicity. Intravenous injection of three doses/13 days, or five doses/20 days, of NPs in mice bearing circulating PC cells and tumor microemboli substantially hindered lung metastasization. Cas9hIL30-PSCA NPs inhibited PC cell proliferation and expression of IL-30 and metastasis drivers, such as CXCR2, CXCR4, IGF1, L1CAM, METAP2, MMP2, and TNFSF10, whereas CDH1 was upregulated. PC-Lung and PC-BM 2-OCs revealed that Cas9hIL30-PSCA NPs suppressed PC cell release of CXCL2/GROß, which was associated with intra-metastatic myeloid cell infiltrates, and of DKK1, OPG, and IL-6, which boosted endothelial network formation and cancer cell migration. Development of a patient-tailored nanoplatform for selective CRISPR-mediated IL-30 gene deletion is a clinically valuable tool against PC progression.

Unleashing the Potential: Designing Antibody-Targeted Lipid Nanoparticles for Industrial Applications with CMC Considerations and Clinical Outlook.

Antibody-targeted lipid nanoparticles (Ab-LNPs) are rapidly gaining traction as multifaceted platforms in precision medicine, adept at delivering a diverse array of therapeutic agents, including nucleic acids and small molecules. This review provides an incisive overview of the latest developments in the field of Ab-LNP technology, with a special emphasis on pivotal design aspects such as antibody engineering, bioconjugation strategies, and advanced formulation techniques. Furthermore, it addresses critical chemistry, manufacturing, and controls (CMC) considerations and thoroughly examines the in vivo dynamics of Ab-LNPs, underscoring their promising potential for clinical application. By seamlessly blending scientific advancements with practical industrial perspectives, this review casts a spotlight on the burgeoning role of Ab-LNPs as an innovative and potent tool in the realm of targeted drug delivery.

Development of PET Radioisotope Copper-64-Labeled Theranostic Immunoliposomes for EGFR Overexpressing Cancer-Targeted Therapy and Imaging.

Combining standard surgical procedures with personalized chemotherapy and the continuous monitoring of cancer progression is necessary for effective NSCLC treatment. In this study, we developed liposomal nanoparticles as theranostic agents capable of simultaneous therapy for and imaging of target cancer cells. Copper-64 (64Cu), with a clinically practical half-life (t1/2 = 12.7 h) and decay properties, was selected as the radioisotope for molecular PET imaging. An anti-epidermal growth factor receptor (anti-EGFR) antibody was used to achieve target-specific delivery. Simultaneously, the chemotherapeutic agent doxorubicin (Dox) was encapsulated within the liposomes using a pH-gradient method. The conjugates of 64Cu-labeled and anti-EGFR antibody-conjugated micelles were inserted into the doxorubicin-encapsulating liposomes via a post-insertion procedure (64Cu-Dox-immunoliposomes). We evaluated the size and zeta-potential of the liposomes and analyzed target-specific cell binding and cytotoxicity in EGFR-positive cell lines. Then, we analyzed the specific therapeutic effect and PET imaging of the 64Cu-Dox-immunoliposomes with the A549 xenograft mouse model. In vivo therapeutic experiments on the mouse models demonstrated that the doxorubicin-containing 64Cu-immunoliposomes effectively inhibited tumor growth. Moreover, the 64Cu-immunoliposomes provided superior in vivo PET images of the tumors compared to the untargeted liposomes. We suggest that nanoparticles will be the potential platform for cancer treatment as a widely applicable theranostic system.

Immunoliposomes As a Promising Antiviral Agent against SARS-CoV-2.

According to the World Health Organization, as of January 3, 2020 to September 13, 2023, there were approximately 23 million confirmed cases of COVID-19 reported in the Russian Federation, about 400 thousand of which were fatal. Considering the high rate of mutation of the RNA-containing virus genome, which inevitably leads to the emergence of new infectious strains (Eris and Pyrola), the search for medicinal antiviral agents remains an urgent task. Moreover, taking into account the actively mutating receptor-binding domain, this task requires fundamentally new solutions. This study proposes a candidate immunoliposomal drug that targets the S protein of SARS-CoV-2 by the monoclonal neutralizing antibody P4A1 and ensures the penetration of a highly active ribonuclease into the virus-infected cell, which degrades, among cellular RNA, viral RNA too. We demonstrate a more than 40-fold increase in the neutralizing activity of the developed drug compared to the free monoclonal neutralizing antibody.

Multifunctional Immunoliposomes Enhance the Immunotherapeutic Effects of PD-L1 Antibodies against Melanoma by Reprogramming Immunosuppressive Tumor Microenvironment.

The immunosuppressive tumor microenvironment (TME) can significantly limit the immunotherapeutic effects of the PD-L1 antibody (aPDL1) by inhibiting the infiltration of CD8+ cytotoxic T cells (CTLs) into the tumor tissues. However, how to reprogram the immunosuppressive TME and promote the infiltration of CTLs remains a huge challenge for aPDL1 to achieve the maximum benefits. Herein, the authors design a multifunctional immunoliposome that encapsulates the adrenergic receptor blocker carvedilol (CAR) and connects the "don't eat me" signal antibody (aCD47) and aPDL1 in series via a reactive oxygen species (ROS)-sensitive linker on the surface. In ROS-enriched immunosuppressive TME, the multifunctional immunoliposome (CAR@aCD47/aPDL1-SSL) can first release the outer aCD47 to block the "do not eat me" pathway, promote the phagocytosis of tumor cells by phagocytic cells, and activate CTLs. Then, the aPDL1 on the liposome surface is exposed to block the PD-1/PD-L1 signaling pathway, thereby inducing CTLs to kill tumor cells. CAR encapsulated in CAR@aCD47/aPDL1-SSL can block the adrenergic nerves in the tumor tissues and reduce their densities, thereby inhibiting angiogenesis in the tumor tissues and reprogramming the immunosuppressive TME. According to the results, CAR@aCD47/aPDL1-SSL holds an effective way to reprogram the immunosuppressive TME and significantly enhance immunotherapeutic efficiency of aPDL1 against the primary cancer and metastasis.

Fibrinspecific liposomes as a potential method of delivery of the thrombolytic preparation streptokinase.

The use of streptokinase (SK) in the clinic is limited by the lack of fibrin-specificity and the short half-life of the drug. We have developed a new dosage form of streptokinase (immunoliposome), which consists of "free" native streptokinase and "bound" encapsulated in liposomes conjugated through carboxylated dextran with fibrin-specific monoclonal antibodies FnI-3C (IgG2 class), in a ratio of 60 and 40%, respectively, and studied their physicochemical properties, pharmacokinetic parameters, and the ability of fibrin-specific liposomes with SK for targeted delivery to fibrin in an in vivo experiment. The obtained immunoliposomes had a hydrodynamic diameter of ~ 140 nm, a zeta potential of - 19.6 mV, and entrapment efficiency of 14.1%. Fluorescent labels bound to immunoliposomes with streptokinase selectively accumulated in model rat vein thrombi at sites containing fibrin in 30 min after injection. Studies of pharmacokinetic parameters showed that the administration of immunoliposomes with streptokinase to rats was accompanied by an increase in the half-life from 1.8 to 24.1 min, the time to reach the maximum concentration from 15 to 30 min, and a decrease in the elimination constant by about 13 times compared with the native streptokinase preparation. Further studies are needed to evaluate the thrombolytic efficacy a new dosage form of streptokinase in experiment in vivo.

The clinical significance of CTC enrichment by GPC3-IML and its genetic analysis in hepatocellular carcinoma.

BACKGROUND: This research was to develop a special method for enriching Circulating tumor cells (CTCs) of Hepatocellular carcinoma (HCC) by Glypican-3 immunoliposomes (GPC3-IML), and to analyze the correlation between the CTCs count and tumor malignancy, as well as to investigate the mutation characteristics of CTC-derived NGS. RESULTS: In this study characterization of physical parameters was performed with the preparation of GPC3-IML. CTCs in peripheral blood of HCC patients were further separated and identified. Immunofluorescence was used to identify CTCs for further counting. By this means, the correlation between CTCs count and clinicopathological features was analyzed, and the genetic mutation characteristics of NGS derived from CTCs were investigated and compared with that of tissue NGS. Results showed that compared with EpCAM and vimentin, GPC-3 had a stronger CTCs separation ability. There was a correlation between "positive" count of CTCs (≥ 5 PV-CTC per 7.5 ml blood) and BCLC stage (P = 0.055). The result of CTC-NGS was consistent with that of tissue-NGS in 60% cases, revealing that KMT2C was a common highly-frequent mutated gene. CONCLUSION: The combination of immunomagnetic separation of CTCs and anti-tumor marker identification technology can be regarded as a new technology of CTCs detection in peripheral blood of patients with HCC. Trial registration EHBHKY2020-k-024. Registered 17 August 2020-Retrospectively registered.

Targeted eradication of gastric cancer stem cells by CD44 targeting USP22 small interfering RNA-loaded nanoliposomes.

AIM: USP22, a member of ubiquitin-specific proteases (USPs), is a well-defined protein that promotes poor prognosis, invasion and metastasis, and also participates in the maintenance of cancer stem cells. USP22 siRNA-loaded nanoliposomes conjugated with CD44 antibodies (USP22-NLs-CD44) were constructed to enhance the therapeutic effect of USP22 siRNA against gastric cancer stem cells. MATERIALS & METHODS: The targeting and therapeutic efficacies of USP22-NLs-CD44 against gastric cancer stem cells were evaluated. RESULTS & CONCLUSION: USP22-NLs-CD44 was demonstrated to be able to effectively deliver USP22 siRNA to CD44+ gastric cancer stem cells, achieving superior therapeutic effects against CD44+ gastric cancer stem cells than nontargeted nanoliposomes. USP22-NLs-CD44 may provide a novel approach to eradicate gastric cancer stem cells in the near future.

High sensitive detection of circulating tumor cell by multimarker lipid magnetic nanoparticles and clinical verifications.

Tumor cells with heterogeneity and diversity can express different markers. At present, positive separation of circulating tumor cells (CTC) taking EpCAM as the marker was used in most cases which could be one-sided, while this study successfully prepared four antibody-modified magnetic immunoliposomes (MIL) by using the self-assembled liposome with antibody derivatives. This study aims to explore the separation efficiency and clinical detection feasibility of single or combined use of MIL with multi-tumor markers on different tumors. Captured CTC were stained with CK-FITC, CD45-PE and DAPI, and fluorescence microscope was used for the observation, analysis and calculation. The result indicated that the CTC number positive rate in blood samples of four different magnetic balls on the same patient could be up to 87.5% in 32 patients with 14 different kinds tumors. While the effect of directly mixed separation by four kinds of magnetic balls was not satisfying. It suggested that the MIL of multi-tumor markers could be a powerful tool for CTC separation in application of tumor screening and prognosis.

The efficacy of anti-VEGF antibody-modified liposomes loaded with paeonol in the prevention and treatment of hypertrophic scars.

The aim of this study was to investigate the efficacy of anti-VEGF antibody-modified Paeonol liposome gels (PAE-BEV-lip gels) in the prevention and treatment of hypertrophic scars (HS). Systematic optimization of the encapsulation process of anti-VEGF antibody-modified Paeonol liposomes (PAE-BEV-lips) was performed using Box-Behnken design with the optimized parameters as follows: SPC concentration of 7.36 mg mL-1; SPC-Chol-PAE:pNP-PEG3000-DOPE:BVE-PEG3000-DOPE ratio of 14:5:4:0.28:0.05, w/w; the hydration temperature of 41 °C; stripping using pH 7.5 sodium dihydrogen phosphate buffer; and ultrasound for 3 min (ultrasound time 2 s, interval 3 s, power 300 W). Using these conditions, the encapsulation efficiency of PAE reached the peak level, i.e. 73.61 ± 2.36%. The PAE-BEV-lips displayed unimodal size-distribution with a mean diameter of (235.7 ± 4.67) nm and a zeta potential of -(5.13 ± 0.25) mV. The investigation of the retention effect PAE-BEV-lip gels revealed a slower transdermal delivery rate, a remarkable dermal retention effect, and superior bioavailability compared to PAE gels and PAE conventional liposome gels (PAE-lip gels). Meanwhile, PAE-BEV-lip gels exhibited definite effects on the prevention and treatment of HS of the rabbit ears. The PAE-BEV-lip gels group showed a lower scar proliferation rate, fewer and looser collagenous fibers and fibromyocytes, more regular chondrocytes, less calcified tissue and fewer inflammatory cells compared to other groups. At the same time, PAE-BEV-lip gels significantly reduced scar hyperplasia index (1.34 ± 0.51) and levels of VEGF, TGF-ß1 and TNF-α (30.90 ± 3.57, 733.2 ± 43.19 and 66.76 ± 2.98 ng·L-1, respectively), compared to the model group (p < .01).

Development and Evaluation of Tri-Functional Immunoliposomes for the Treatment of HER2 Positive Breast Cancer.

PURPOSE: Trastuzumab combined with Doxorubicin (DOX) demonstrates significant clinical activity in human epidermal growth factor receptor-2 (HER2)-positive breast cancer (BC). However, emergence of treatment resistance and trastuzumab associated cardiotoxicity remain clinical challenges. In an effort to improve patient outcome, we have developed and evaluated novel tri-functional immunoliposomes (TFIL) that target HER2-receptors on BC cells and CD3-receptors on T-lymphocytes, and deliver DOX. METHODS: Trastuzumab (anti-HER2) and OKT-3 (anti-CD3) antibodies were conjugated to liposomes using a micelle-transfer method. Cytotoxicity of targeted immunoliposomes loaded with DOX was examined in vitro on HER2-positive BC cells (BT474), with peripheral blood monocytic cells (PBMC) as immune effector cells. RESULTS: TFIL demonstrated high antibody-liposome conjugation ratios (100-130 µg protein/µmol phospholipid) and cargo capacity (0.21 mol:mol drug:lipid), highly efficient DOX loading (>90%), a particle size favorable for extended circulation (~150 nm), and good stability (up to 3 months at 4°C). In the presence of PBMCs, TFIL showed complete killing of BT474 cells, and were superior to mono-targeted trastuzumab-bearing liposomes, non-targeted liposomes, and free Trastuzumab and DOX. CONCLUSIONS: Novel anti-HER2xCD3 + DOX TFIL show promise as a means to both engage immune cells against HER2 positive breast cancer cells and deliver chemotherapy, and have the potential to improve clinical outcomes.

Anti-GD2 Immunoliposomes for Targeted Delivery of the Survivin Inhibitor Sepantronium Bromide (YM155) to Neuroblastoma Tumor Cells.

PURPOSE: Sepantronium bromide (YM155) is a hydrophilic quaternary compound that cannot be administered orally due to its low oral bioavailability; it is furthermore rapidly eliminated via the kidneys. The current study aims at improving the pharmacokinetic profile of YM155 by its formulation in immunoliposomes that can achieve its enhanced delivery into tumor tissue and facilitate uptake in neuroblastoma cancer cells. METHODS: PEGylated YM155 loaded liposomes composed of DPPC, cholesterol and DSPE-PEG2000 were prepared via passive film-hydration and extrusion method. Targeted (i.e. immuno-)liposomes were prepared by surface functionalization with SATA modified monoclonal anti-disialoganglioside (GD2) antibodies. Liposomes were characterized based on their size, charge, antibody coupling and YM155 encapsulation efficiency, and stability. Flow cytometry analysis and confocal microscopy were performed on IMR32 and KCNR neuroblastoma cell lines. The efficacy of developed formulations were assessed by in-vitro toxicity assays. A pilot pharmacokinetic analysis was performed to assess plasma circulation and tumor accumulation profiles of the developed liposomal formulations. RESULTS: YM155 loaded immunoliposomes had a size of 170 nm and zeta potential of -10 mV, with an antibody coupling efficiency of 60% andYM155 encapsulation efficiency of14%. Targeted and control liposomal formulations were found to have similar YM155 release rates in a release medium containing 50% serum. An in-vitro toxicity study on KCNR cells showed less toxicity for immunoliposomes as compared to free YM155. In-vivo pharmacokinetic evaluation of YM155 liposomes showed prolonged blood circulation and significantly increased half-lives of liposomal YM155 in tumor tissue, as compared to a bolus injection of free YM155. CONCLUSIONS: YM155 loaded immunoliposomes were successfully formulated and characterized, and initial in-vivo results show their potential for improving the circulation time and tumor accumulation of YM155.

Drug delivery to the human and mouse uterus using immunoliposomes targeted to the oxytocin receptor.

BACKGROUND: The ability to provide safe and effective pharmacotherapy during obstetric complications, such as preterm labor or postpartum hemorrhage, is hampered by the systemic toxicity of therapeutic agents leading to adverse side effects in the mother and fetus. Development of novel strategies to target tocolytic and uterotonic agents specifically to uterine myocytes would improve therapeutic efficacy while minimizing the risk of side effects. Ligand-targeted liposomes have emerged as a reliable and versatile platform for targeted drug delivery to specific cell types, tissues or organs. OBJECTIVE: Our objective was to develop a targeted drug delivery system for the uterus utilizing an immunoliposome platform targeting the oxytocin receptor. STUDY DESIGN: We conjugated liposomes to an antibody that recognizes an extracellular domain of the oxytocin receptor. We then examined the ability of oxytocin receptor-targeted liposomes to deliver contraction-blocking (nifedipine, salbutamol and rolipram) or contraction-enhancing (dofetilide) agents to strips of spontaneously contracting myometrial tissue in vitro (human and mouse). We evaluated the ability of oxytocin receptor-targeted liposomes to localize to uterine tissue in vivo, and assessed if targeted liposomes loaded with indomethacin were capable of preventing lipopolysaccharide-induced preterm birth in mice. RESULTS: Oxytocin receptor-targeted liposomes loaded with nifedipine, salbutamol or rolipram consistently abolished human myometrial contractions in vitro, while oxytocin receptor-targeted liposomes loaded with dofetilide increased contraction duration. Nontargeted control liposomes loaded with these agents had no effect. Similar results were observed in mouse uterine strips. Following in vivo administration to pregnant mice, oxytocin receptor-targeted liposomes localized specifically to the uterine horns and mammary tissue. Targeting increased localization to the uterus 7-fold. Localization was not detected in the maternal brain or fetus. Targeted and nontargeted liposomes also localized to the liver. Oxytocin receptor-targeted liposomes loaded with indomethacin were effective in reducing rates of preterm birth in mice, whereas nontargeted liposomes loaded with indomethacin had no effect. CONCLUSION: Our results demonstrate that oxytocin receptor-targeted liposomes can be used to either inhibit or enhance human uterine contractions in vitro. In vivo, the liposomes localized to the uterine tissue of pregnant mice and were effective in delivering agents for the prevention of inflammation-induced preterm labor. The potential clinical advantage of targeted liposomal drug delivery to the myometrium is reduced dose and reduced toxicity to both mother and fetus.

Targeted tumor MRI with gadobutrol-loaded anti-HER2 immunoliposomes.

Background Immunoliposomes have been used to deliver MR contrast agents to cancer tissue by targeting tumor associated antigens, thus enabling the visualization of biological processes at the cellular level. Purpose To develop and evaluate the feasibility of specific HER2 targeted liposomal MR contrast agent. Material and Methods Gd-loaded anti-HER2 immunolipomes (Gd-ILs) and non-targeted PEGylated liposomes (Gd-NTLs) were prepared and characterized. Tumor bearing animals were randomized into three groups: Gd- ILs, Gd- NTLs and gadobutrol. Animals were imaged prior and 5, 15, 60, 120 and 180 min after i.v. injection of different contrast agents. The signal intensity enhancement percentage, signal- to- noise ratio and contrast- to -noise ratio was used to qualify tumor enhancement of different groups. After imaging, tumors were excised for histological examination. Results In vivo dynamic MR images, the specific targeted contrast agent bound to tumor tissue and result in a gradual and persisting enhancement for at least 3 hours in mice bearing tumor xenografts, reaching a maximum of 87.7% enhancement after 120 min post-injection. Gd-ILs demonstrated superior tumor enhancement over control non target contrast agent and gadobutrol in HER2 overexpressing tumors at 60, 120 and 180 min post- injection. The SNR and CNR of Gd-ILs in the tumors were significantly greater than that of Gd-NTLs at 60, 120, 180 min post- injection. Conclusion The results indicate the feasibility of Gd-ILs providing prolonged circulation, specific tumor enhancement and cancer cell recognition as targeted contrast agent.

Construction and evaluation of pH-sensitive immunoliposomes for enhanced delivery of anticancer drug to ErbB2 over-expressing breast cancer cells.

1,5-Dihexadecyl N,N-diglutamyl-lysyl-L-glutamate (GGLG) liposomes were previously developed to enhance drug delivery efficiency in tumor cells owing to its pH-responsive properties. Herein, we report the modification of GGLG liposomes by conjugating a Fab' fragment of an ErbB2 antibody to the terminus of PEG (polyethylene glycol)-lipid (Fab'-GGLG liposomes). The conjugation of Fab' fragments did not affect the antibody activity, drug (doxorubicin, DOX) encapsulation efficiency, stability during storage or pH-sensitivity. However, the binding affinity of Fab'-GGLG liposomes was enhanced to ErbB2-overexpressing HCC1954 cells specifically, and the cell association increased 10-fold in comparison to GGLG liposomes. Consequently, intracellular DOX delivery was enhanced, with an increased cytotoxicity in HCC1954 cells (i.e., IC50 of 1.17 and 3.08 µg/mL for Fab'-GGLG-DOX and GGLG-DOX liposomes, respectively). Further, a significantly enhanced tumor growth inhibition was obtained in an ErbB2-overexpressing breast cancer-bearing mouse model. Therefore, a potent anticancer drug delivery system was constructed by the immunological modification of pH-sensitive liposomes.

Characterization and in vitro evaluation of nimotuzumab conjugated with cisplatin-loaded liposomes.

In this paper, we report the conjugation of the humanized monoclonal antibody nimotuzumab with cisplatin-loaded liposomes and the in vitro evaluation of its affinity for tumor cells. The conjugation procedure was performed through derivatization of nimotuzumab with N-succinimidyl S-acetylthioacetate (SATA) followed by a covalent attachment with maleimide groups at the end of PEG-DSPE chains located at the membrane of pre-formed liposomes. Confocal microscopy was performed to evaluate the immunoliposome affinity for EGFR antigens from human epidermoid carcinoma (A-431) and normal lung (MRC-5) cell lines. Results showed that the procedures implemented in this work do not affect the capability of the nimotuzumab-immunoliposomes to recognize the tumor cells, which overexpress the EGFR antigens.

Effect of lipid composition on incorporation of trastuzumab-PEG-lipid into nanoliposomes by post-insertion method: physicochemical and cellular characterization.

CONTEXT: Anti-HER2 immunoliposomes are promising nanotechnology based systems for active targeting of breast tumors, which depends on the amount of incorporated antibody. OBJECTIVE/AIM: In this work, we investigated the possible effect of lipid composition on the incorporation of trastuzumab-PEG-PE micelles into nanoliposomes and on their subsequent specific cellular targeting. MATERIALS AND METHODS: Trastuzumab (anti-HER2 monoclonal antibody) was monothiolated and conjugated to maleimide-PEG-PE micelles. Liposomes of different lipid compositions were prepared by the thin layer hydration. Trastuzumab-PEG-PE micelles were incorporated into the liposomes by the post-insertion method. The percentage of lipid mixing was determined based on fluorescence resonance energy transfer. Cellular binding and uptake of rhodamine-labeled immunoliposomes were studied in SKBR-3 (HER2(+++)) and MCF-7 (HER2(+)) cells. Also, antitumor cell activity of the immunoliposomes was compared to free trastuzumab and the liposomes. RESULTS: The lipid mixing of trastuzumab-PEG-PE micelles depended on the liposome composition. The immunoliposomes containing DPPC, cholesterol and PEG-PE showed prominent lipid mixing. The lipid mixing was consistent with the cell binding results which showed an efficient and specific binding of the immunoliposomes to SKBR-3 cells. Antitumor cell activity of the immunoliposomes in SKBR-3, unlike MCF-7 cells, depended on the content of trastuzumab. DISCUSSION: Cholesterol and PEG-PE in the liposome composition are prerequisites for a successful lipid mixing due to their ability to facilitate fusion. The higher lipid mixing results in higher antibody incorporation and consequently higher targeted cell binding. CONCLUSIONS: The lipid mixing depends on the liposome composition, which reflects targeted cell binding of the immunoliposomes.

Liposomes Coloaded with Elacridar and Tariquidar To Modulate the P-Glycoprotein at the Blood-Brain Barrier.

This study prepared three liposomal formulations coloaded with elacridar and tariquidar to overcome the P-glycoprotein-mediated efflux at the blood-brain barrier. Their pharmacokinetics, brain distribution, and impact on the model P-glycoprotein substrate, loperamide, were compared to those for the coadministration of free elacridar plus free tariquidar. After intravenous administration in rats, elacridar and tariquidar in conventional liposomes were rapidly cleared from the bloodstream. Their low levels in the brain did not improve the loperamide brain distribution. Although elacridar and tariquidar in PEGylated liposomes exhibited 2.6 and 1.9 longer half-lives than free elacridar and free tariquidar, respectively, neither their Kp for the brain nor the loperamide brain distribution was improved. However, the conjugation of OX26 F(ab')2 fragments to PEGylated liposomes increased the Kps for the brain of elacridar and tariquidar by 1.4- and 2.1-fold, respectively, in comparison to both free P-gp modulators. Consequently, the Kp for the brain of loperamide increased by 2.7-fold. Moreover, the plasma pharmacokinetic parameters and liver distribution of loperamide were not modified by the PEGylated OX26 F(ab')2 immunoliposomes. Thus, this formulation represents a promising tool for modulating the P-glycoprotein-mediated efflux at the blood-brain barrier and could improve the brain uptake of any P-glycoprotein substrate that is intended to treat central nervous system diseases.

Immunoliposomes for Targeted Delivery of an Antifibrotic Drug.

Excessive extracellular matrix formation in organs and tissues arises from an imbalance between the synthesis and degradation of matrix proteins, especially collagen. This condition interferes with proper wound healing and regeneration, and to date, no specific treatment is available. In the present study, we propose a targeted drug delivery system consisting of cell-specific immunoliposomes (ILs) loaded with deferoxamine (DFO) as an antifibrotic drug. ILs were functionalized with polyethylene glycol (PEG) to improve the steric stability and prolong their half-life. In addition, a single-chain Fv (scFv) antibody fragment that specifically targets fibroblast activation protein (FAP) was incorporated. An in vitro fibrosis model was employed to test this construct. This model consisted of highly activated pro-fibrotic fibroblasts with 2- to 6-fold induction of selected fibrosis markers: cell/matrix deposited collagen I, total soluble collagen, and α smooth muscle actin. The activation was accompanied by a significant and cell-specific elevation of FAP expression and activity, thereby confirming that FAP is an adequate target for antifibrotic drug delivery. Purified anti-FAP scFv was shown to bind specifically to these cells without influencing the FAP enzymatic activity. DFO was demonstrated to have a dose-dependent antifibrotic activity as quantified by collagen deposition. Specific binding and intracellular uptake of DiI-labeled ILs into the activated fibroblasts were shown by flow cytometry and microscopy. Finally, DFO-loaded ILs targeted to FAP caused a significant reduction in the collagen deposition, whereas no effect was observed using liposomes that lacked the targeting antibody fragment. These results suggest that the FAP-specific scFv-conjugated liposomes have considerable potential for cell-specific targeting applicable as a therapy for excessive collagen deposition during fibrosis. In general, through liposome encapsulation, bioactive molecules, such as DFO, that have broad effects and poor cell penetration can be converted into cell-specific composites for targeted drug delivery.

Functionalization of liposomes: microscopical methods for preformulative screening.

The development of smart delivery systems able to deliver and target a drug to the site of action is one of the major challenges in the field of pharmaceutical technology. The surface modification of nanocarriers, such as liposomes, is widely investigated either for increasing the blood circulation time (by pegylation) or for interacting with specific tissues or cells (by conjugation of a selective ligand as a monoclonal antibody, mAb). Microscopical analysis thereby is a useful approach to evaluate the morphology and the size owing to resolution and versatility in defining either surface modification or the architecture and the internal structure of liposomes. This contribution aims to connect the outputs obtained by transmission electron (TEM) and atomic force (AFM) microscopical techniques for identifying the modifications on the liposomal surface. To reach this objective, we prepared liposomes applying two different pegylation technologies and further modifying the surface by mAb conjugation. This work demonstrates the feasibility to apply the combined approach (TEM and AFM analysis) in the evaluation of the efficacy of a surface engineering process.