Tumor targeting of functionalized lipid nanoparticles: Assessment by in vivo fluorescence imaging

Abstract

Lipid nanoparticles (LNP) coated by a poly(oxyethylene) polymer have been manufactured from low cost and human use-approved materials, by an easy, robust, and up-scalable process. The incorporation in the formulation of maleimide-grafted surfactants allows the functionalization of the lipid cargos by targeting ligands such as the cRGD peptide binding to α_vβ₃ integrin, a well-known angiogenesis biomarker. LNP are able to encapsulate efficiently lipophilic molecules such as a fluorescent dye, allowing their in vivo tracking using fluorescence imaging. In vitro study on HEK293(β3) cells over-expressing the α_vβ₃ integrins demonstrates the functionalization, specific targeting, and internalization of cRGD-functionalized LNP in comparison with LNP-cRAD or LNP-OH used as negative controls. Following their intravenous injection in Nude mice, LNP-cRGD can accumulate actively in slow-growing HEK293(β3) cancer xenografts, leading to tumor over skin fluorescence ratio of 1.53 ± 0.07 (n = 3) 24 h after injection. In another fast-growing tumor model (TS/A-pc), tumor over skin fluorescence ratio is improved (2.60 ± 0.48, n = 3), but specificity between the different LNP functionalizations is no more observed. The different results obtained for the two tumor models are discussed in terms of active cRGD targeting and/or passive nanoparticle accumulation due to the Enhanced Permeability and Retention effect.

Introduction

The use of effective cancer chemotherapy drugs is mainly hindered by their low aqueous solubility, which complicates their administration, and their high toxicity, necessary in regards to cancer cells but detrimental and the origin of severe side effects for healthy tissues. A wide range of pharmaceutical nanocarriers, such as polymeric or lipid nanoparticles or micelles [1], [2], [3] and even inorganic nanoparticles [4], have been screened for reducing the administrated doses and enhancing the in vivo accumulation of therapeutic agents into the areas to be cured. Polymeric nanomaterials are very often composed of FDA-approved biodegradable polymers, such as poly(lactic acid) (PLA), poly(glycolic acid) (PGA), and poly(ε-caprolactone) (PCL). However, their structure and therefore their synthesis process have to be tailored for each specific application. Lipid materials are more versatile and present the advantage of a lower intrinsic toxicity, because of their compositions similar or close to those of physiological lipids.

Numerous lipid nanocarriers with different structures have been studied for therapy or imaging [5], [6], [7]: micelles [8], lipoproteins [9], [10], liposomes [11], micro-emulsions [12] and nanoemulsions [13], [14], solid-lipid nanoparticles (SLN) [6], [15], [16] and nanocapsules [17]. Because of the limited size of their core, micelles cannot store high drug payload. Lipoproteins must be isolated from fresh plasma or designed from synthetic peptides [18]. Due to their phospholipid bilayer and hydrophilic cavity, liposomes display outstanding properties for the encapsulation of hydrophilic/amphiphilic therapeutic agents. However, they are not optimized for the encapsulation of high concentrations of lipophilic drugs. They are not stable enough for being stored in injection-ready formulations for long durations, and their use usually requires the reconstitution of the solution just before injection to the patient. Lipid core nanocarriers present the advantage of a higher volume reservoir for lipophilic drug encapsulation. Nanocapsules have a liquid-lipid core encapsulated within a solid polymeric or phospholipid shell [17]. Micro-emulsions (self-assembled, thermodynamically stable) [12] and nanoemulsions (thermodynamically instable, requiring energy for their processing) [13], [14] have a liquid core, whereas solid-lipid nanoparticles (SLN) have in principle a solid one [6], [15], [16]. However, the frontiers between the different categories of lipid particles are not so well defined. As an example, super-cooled liquid state has been observed in different SLN formulations. The solid crystalline core of SLN can present several drawbacks, such as problems of reproducibility in the particle growth, possibility of polymorphic transitions, which can induce drug expulsion during storage, and low drug incorporation capacities due to the crystalline structure [19]. Therefore, the concept of “nanostructured” lipid carriers has been introduced by Müller et al. [5]. The idea is to obtain lipid nanoparticles with a solid but not crystalline core, in order to improve the efficiency and stability of drug encapsulation.

In this manuscript, we report the design and in vivo bio-distribution of new lipid nanoparticles (LNP), which lipid core is composed of a mixture of oil and wax, solid at room temperature for improved stability and liquid at body temperature (≈37–40 °C melting point). The core of the LNP therefore provides a reservoir suitable for the encapsulation and controlled release of lipophilic drugs and molecules. In order to demonstrate the potential of these new nanocarriers and to assess their in vivo fate, whole body non-invasive fluorescence imaging (FLI) in mice is used. We have recently reported the efficient loading of a lipophilic near infrared dye, DiD, within the lipid core of the LNP and the benefits of encapsulation on the dye properties (improved fluorescence quantum yield and reduced photobleaching) [20]. FLI now allows to scan in real time the bio-distribution of near infrared dye-labeled molecules (μM payloads) in whole body live mice in a few seconds with a few millimeters resolution [21], [22], [23]. FLI is a fast, low cost, sensitive, non-ionizing, non-invasive, and easy-handled screening technique.

Using DiD-loaded nanoparticles, the bio-distribution of LNP decorated by the cRGD peptide able to target α_vβ₃ integrins has therefore been studied in vivo in two mice models using the FLI technique. The functionalization of nanocargos in order to improve their vectorization and targeting properties has been a major issue explored to improve therapy and diagnostics efficiencies in the last three decades [1], [2], [3]. α_vβ₃ Integrins are receptors over-expressed on the angiogenic vessels grown during tumor development [24], [25]. Moreover, these membrane receptors are also expressed in 25% of human cancer cells of different types (melanoma, glioblastoma, ovarian, breast cancer) [24], [25]. They therefore constitute targets of choice for favoring the specific binding and uptake in malignant cells of nanoparticles loaded with a drug or contrast agent. The well-known triad peptide sequence RGD (Arg-Gly-Asp), which recognizes the α_vβ₃ integrins has been identified 20 years ago [26], and its cyclic form c[-RGDfK-] designed from the peptides developed by Kessler’s group provides easy conjugation to imaging and/or therapeutic moieties [24], [27], [28]. Grafting onto the LNP of the cRGD targeting ligand, the cRAD peptide negative control or a non-targeting –OH group is hereafter assessed by in vitro test on HEK293(β3) cultured cells, before the functionalized LNP are injected intravenously in two mice models, HEK293(β3) and TS/A-pc. The HEK293(β3) cell line (human embryonic kidney) has been genetically modified to strongly express α_vβ₃ integrins and has been successfully used as a study model for a few years [29], [30], [31]. TS/A-pc (mice mammary carcinoma cells) is another model which express naturally α_vβ₃ integrins [32]. Whenever injected subcutaneously in a Nude mouse model, TS/A-pc cells will grow faster (10 days) than HEK293(β3) cells (6 weeks) before reaching the size at which tumor imaging is performed. It is important whenever designing a new fluorescent probe or a drug delivery vector to demonstrate its potentialities in different animal models. In this study, LNP-cRGD-specific targeting is studied in comparison with negative control LNP-cRAD and non-targeted LNP-OH in the two different mice models, HEK293(β3) and TS/A-pc, which can be targeted using the same ligand, but differ in their growing process.