Louis van Bloois

Synovial macrophage depletion with clodronate-containing liposomes in rheumatoid arthritis

OBJECTIVE To assess whether intraarticular (IA) administration of clodronate liposomes results in local macrophage depletion in patients with rheumatoid arthritis (RA). Primary goals were to address both the immunohistologic and potential toxic effects of this approach. Moreover, the correlation between immunohistologic findings and clinical assessments of disease activity and cartilage damage were assessed. METHODS An open study was conducted in consecutive RA patients who were scheduled for knee joint replacement in our department. Synovial biopsy tissue was obtained from the knee joint at 2 weeks before and at the time of surgery. This protocol was controlled for safety and immunohistologic concordance in 6 patients. One week before surgery, 10 patients received a single IA dose of clodronate liposomes. Staining of synovial tissue for cell markers (CD68, CD14, CD3, CD38) and adhesion molecules (vascular cell adhesion molecule 1 [VCAM-1], intercellular adhesion molecule 1 [ICAM-1]) was assessed by 2 blinded observers. Local and systemic parameters of disease activity were measured before each intervention. Cartilage damage was scored using standard radiologic techniques at baseline and during surgery. RESULTS A single IA dose of clodronate liposomes significantly reduced the number of CD68-positive cells (P = 0.005) and the expression of ICAM-1 and VCAM-1 in the synovial lining (P = 0.013 and P = 0.039, respectively). The intervention did not affect fibroblast-like synoviocytes, T cells, or plasma cells. No immunohistologic changes were observed in the control group. The procedure was well tolerated. The levels of ICAM-1 and VCAM-1 in the sublining layers correlated with the extent of macroscopic synovitis (P < 0.0005 and P < 0.005, respectively). The expression of ICAM-1 and CD14 in the sublining correlated with the levels of C-reactive protein (P < 0.0005 and P < 0.01, respectively). Cartilage destruction was correlated only with the expression of CD68 in the sublining (P = 0.02). CONCLUSION A single IA administration of clodronate liposomes leads to macrophage depletion and decreased expression of adhesion molecules in the synovial lining in patients with longstanding RA. The procedure is well tolerated, and its therapeutic potential is currently under investigation. The expression of adhesion molecules in the sublining layers reflects ongoing inflammation.

Improving solubility and chemical stability of natural compounds for medicinal use by incorporation into liposomes

Natural bioactive compounds have been studied for a long time for their chemopreventive and therapeutic potential in several chronic inflammatory diseases, including cancer. However, their physicochemical properties generally result in poor chemical stability and lack of in vivo bioavailability. Very few human clinical trials have addressed absorption, distribution, metabolism, and excretion of these compounds in relation to efficacy. This limits the use of these valuable natural compounds in the clinic. In this study, we examined caffeic acid (derivatives), carvacrol (derivatives), thymol, pterostilbene (derivatives), and N-(3-oxo-dodecanoyl)-l-homoserine lactone. These are natural compounds with strong anti-inflammatory properties derived from plants and bacteria. However, these compounds have poor water solubility or are chemically unstable. To overcome these limitations we have prepared liposomal formulations. Our results show that lipophilic 3-oxo-C(12)-homoserine lactone and stilbene derivatives can be loaded into liposomal lipid bilayer with efficiencies of 50-70%. Thereby, the liposomes solubilize these compounds, allowing intravenous administration without use of solvents. When compounds could not be loaded into the lipid bilayer (carvacrol and thymol) or are rapidly extracted from the liposomes in the presence of serum albumin (3-oxo-C(12)-homoserine lactone and pterostilbene derivatives), derivatization of the compound into a water-soluble prodrug was shown to improve loading efficiency and encapsulation stability. The phosphate forms of carvacrol and pterostilbene were loaded into the aqueous interior of the liposomes and encapsulation was unaffected by the presence of serum albumin. Chemical instability of resveratrol was improved by liposome-encapsulation, preventing inactivating cis-trans isomerization. For caffeic acid, liposomal encapsulation did not prevent oxidation into a variety of products. Still, by derivatization into a phenyl ester, the compound could be stably encapsulated without chemical degradation. Despite the instability of liposome-association of 3-oxo-C(12)-homoserine lactone and resveratrol, intravenous administration of these compounds inhibited tumor growth for approximately 70% in a murine tumor model, showing that simple solubilization can have important therapeutic benefits.

Multimodal clinical imaging to longitudinally assess a nanomedical anti-inflammatory treatment in experimental atherosclerosis.

Atherosclerosis is an inflammatory disease causing great morbidity and mortality in the Western world. To increase the anti-inflammatory action and decrease adverse effects of glucocorticoids (PLP), a nanomedicinal liposomal formulation of this drug (L-PLP) was developed and intravenously applied at a dose of 15 mg/kg PLP to a rabbit model of atherosclerosis. Since atherosclerosis is a systemic disease, emerging imaging modalities for assessing atherosclerotic plaque are being developed. (18)F-Fluoro-deoxy-glucose positron emission tomography and dynamic contrast enhanced magnetic resonance imaging, methods commonly used in oncology, were applied to longitudinally assess therapeutic efficacy. Significant anti-inflammatory effects were observed as early as 2 days that lasted up to at least 7 days after administration of a single dose of L-PLP. No significant changes were found for the free PLP treated animals. These findings were corroborated by immunohistochemical analysis of macrophage density in the vessel wall. In conclusion, this study evaluates a powerful two-pronged strategy for efficient treatment of atherosclerosis that includes nanomedical therapy of atherosclerotic plaques and the application of noninvasive and clinically approved imaging techniques to monitor delivery and therapeutic responses. Importantly, we demonstrate unprecedented rapid anti-inflammatory effects in atherosclerotic lesions after the nanomedical therapy.

Glycan-modified liposomes boost CD4 + and CD8 + T-cell responses by targeting DC-SIGN on dendritic cells

Cancer immunotherapy requires potent tumor-specific CD8(+) and CD4(+) T-cell responses, initiated by dendritic cells (DCs). Tumor antigens can be specifically targeted to DCs in vivo by exploiting their expression of C-type lectin receptors (CLR), which bind carbohydrate structures on antigens, resulting in internalization and antigen presentation to T-cells. We explored the potential of glycan-modified liposomes to target antigens to DCs to boost murine and human T-cell responses. Since DC-SIGN is a CLR expressed on DCs, liposomes were modified with DC-SIGN-binding glycans Lewis (Le)(B) or Le(X). Glycan modification of liposomes resulted in increased binding and internalization by BMDCs expressing human DC-SIGN. In the presence of LPS, this led to 100-fold more efficient presentation of the encapsulated antigens to CD4(+) and CD8(+) T-cells compared to unmodified liposomes or soluble antigen. Similarly, incubation of human moDC with melanoma antigen MART-1-encapsulated liposomes coated with Le(X) in the presence of LPS led to enhanced antigen-presentation to MART-1-specific CD8(+) T-cell clones. Moreover, this formulation drove primary CD8(+) T-cells to differentiate into high numbers of tetramer-specific, IFN-γ-producing effector T-cells. Together, our data demonstrate the potency of a glycoliposome-based vaccine targeting DC-SIGN for CD4(+) and CD8(+) effector T-cell activation. This approach may offer improved options for treatment of cancer patients and opens the way to in situ DC-targeted vaccination.

Possibilities and limitations of current technologies for quantification of biological extracellular vesicles and synthetic mimics

Nano-sized extracelullar vesicles (EVs) released by various cell types play important roles in a plethora of (patho)physiological processes and are increasingly recognized as biomarkers for disease. In addition, engineered EV and EV-inspired liposomes hold great potential as drug delivery systems. Major technologies developed for high-throughput analysis of individual EV include nanoparticle tracking analysis (NTA), tunable resistive pulse sensing (tRPS) and high-resolution flow cytometry (hFC). Currently, there is a need for comparative studies on the available technologies to improve standardization of vesicle analysis in diagnostic or therapeutic settings. We investigated the possibilities, limitations and comparability of NTA, tRPS and hFC for analysis of tumor cell-derived EVs and synthetic mimics (i.e. differently sized liposomes). NTA and tRPS instrument settings were identified that significantly affected the quantification of these particles. Furthermore, we detailed the differences in absolute quantification of EVs and liposomes using the three technologies. This study increases our understanding of possibilities and pitfalls of NTA, tRPS and hFC, which will benefit standardized and large-scale clinical application of (engineered) EVs and EV-mimics in the future.

Biosynthetically lipid-modified human scFv fragments from phage display libraries as targeting molecules for immunoliposomes

A human anti‐CD22 single chain (sc) Fv antibody fragment from a synthetic phage antibody display library was biosynthetically lipid‐tagged by using Escherichia coli lipoprotein sequences. The purified anti‐CD22 scFv lipoprotein was incorporated into liposomes by detergent dilution. Anti‐CD22 immunoliposomes were shown to bind specifically in a dose‐ and time‐dependent manner to CD22+ cell lines and CD22+ B‐lymphocytes present in freshly isolated samples of blood mononuclear cells. The immunoliposomes were demonstrated to accumulate in intracellular compartments. Biosynthetically lipid‐tagged human scFv antibody fragments isolated from phage display libraries may facilitate the construction of immunoliposomes with improved properties.

The interaction of cytostatic drugs with adsorbents in aqueous media. The potential implications for liposome preparation

A new method that involves the use of the cation exchange resin Dowex 50W-X4 to remove non-encapsulated drugs from liposome dispersions was investigated. Cytostatic drugs widely varying in their molecular structure can be removed from aqueous solutions by Dowex 50W-X4. The applicability of the resin to separate free from liposome-bound drugs was illustrated for a number of cytostatic drugs (cisplatin, doxorubicin, vincristine). The technique presented allows for a rapid, efficient and convenient procedure for the free drug removal from liposome dispersions without dilution of the liposomal preparation. Studies with liposome-encapsulated drugs will be facilitated by the use of this method, since it avoids many of the problems introduced by conventional methods as dialysis, gel filtration and centrifugation/washing. To elucidate the interaction mechanism of doxorubicin with Dowex 50W-X4, alternative adsorbents were studied for their doxorubicin binding properties. In the adsorption process of doxorubicin onto Dowex 50W-X4 both electrostatic (ion exchange) and hydrophobic effects play a role. The results indicate that hydrophobic contributions to the interaction are responsible for the high resistance offered by the binding forces against desorption of adsorbed doxorubicin. For other adsorbents the interactions are either mainly of an electrostatic or a hydrophobic nature.

Comparison of 24 different adjuvants for inactivated HIV-2 split whole virus as antigen in mice. Induction of titres of binding antibodies and toxicity of the formulations

The objective of this study was the comparison of the quantity and quality of the humoral immune response against inactivated whole human immunodeficiency virus type 2. Twenty-four different adjuvants were tested. The toxicity of these preparations was determined. Due to the large number of experiments, the animal model used was the NMRI mouse. Sera were assayed for the presence of antibodies by ELISA and Western blot. The toxicity of the adjuvants was determined by observing lethal side-effects occurring within two days after injection of the immunogenic preparations. The results show that polymethylmethacrylate nanoprticles were the best overall adjuvant, inducing the highest titres of antibodies compared with other adjuvants as well as producing a significantly better immune response against a number of individual HIV-2 antigens without any observable toxic side-effects. However, this adjuvant did not induce antibodies against the outer envelope protein gp120, whereas such antibodies were induced by aluminium compounds, Freunds complete and incomplete adjuvants, and fumed silica (Aerosil). This result suggests the necessity of using vaccines with two or more different adjuvants in order to induce the required immune response against physically different antigens.

Contribution of Classic and Alternative Effector Pathways in Peanut-Induced Anaphylactic Responses

Food allergy affects approximately 5% of children and is the leading cause of hospitalization for anaphylactic reactions in westernized countries. However, the pathways of anaphylaxis in food allergy are still relatively unknown. We investigated the effector pathways of allergic and anaphylactic responses of different strains of mice in a clinical relevant model of peanut allergy. C3H/HeOuJ, C57BL/6 and BALB/c mice were sensitized by intragastric peanut extract and challenged by intragastric or intraperitoneal injection of peanut. Peanut-specific T cell responses, IgE, IgG1 and IgG2a and mucosal mast cell degranulation were induced to different extent in C3H/HeOuJ, C57BL/6 and BALB/c mice. Interestingly, anaphylactic symptoms after systemic challenge were highest in C3H/HeOuJ followed by C57BL/6 but were absent in BALB/c mice. Mechanistic studies showed that the food allergic systemic anaphylaxis was dependent on platelets, FcRγ and mast cells, and partially dependent on platelet activating factor and monocytes/macrophages, depending on mouse strain. These data demonstrate that in three mouse strains, components of the classic and alternative anaphylactic cascade are differently expressed, leading to differential outcomes in parameters of allergic disease and food induced systemic anaphylaxis.

Betulinic acid delivered in liposomes reduces growth of human lung and colon cancers in mice without causing systemic toxicity

Betulinic acid (BetA) is a plant-derived pentacyclic triterpenoid with potent anticancer capacity that targets the mitochondrial pathway of apoptosis. BetA has a broad efficacy in vitro against prevalent cancer types, including lung, colorectal, prostate, cervix and breast cancer, melanomas, neuroblastomas, and leukemias. The cytotoxic effects of the compound against healthy cells are minimal, rendering BetA a promising potential anticancer drug. However, because of the weak hydrosolubility of BetA, it has been difficult to study its efficacy in vivo and a pharmaceutical formulation is not yet available. We report the development of a liposome formulation of BetA and show its successful application in mice. Large liposomes, assembled without cholesterol to reduce their rigidity, efficiently incorporated BetA. Nude mice xenografted with human colon and lung cancer tumors were treated intravenously with the BetA-containing liposomes. Tumor growth was reduced to more than 50% compared with the control treatment, leading to an enhanced survival of the mice. Oral administration of the liposomal formulation of BetA also slowed tumor growth. Any signs of systemic toxicity caused by BetA treatment were absent. Thus, liposomes are an efficient formulation vehicle for BetA, enabling its preclinical development as a nontoxic compound for the treatment of cancers.