The chick embryo and its chorioallantoic membrane (CAM) for the in vivo evaluation of drug delivery systems

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Advanced Drug Delivery Reviews 59 (2007) 1162 – 1176 www.elsevier.com/locate/addr

The chick embryo and its chorioallantoic membrane (CAM) for the in vivo evaluation of drug delivery systems ☆ Angelica Vargas, Magali Zeisser-Labouèbe, Norbert Lange, Robert Gurny, Florence Delie ⁎ Department of Pharmaceutics and Biopharmaceutics, School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, 30, Quai Ernest Ansermet, CH-1211 Geneva 4, Switzerland Received 9 February 2007; accepted 20 April 2007 Available online 16 August 2007

Abstract Mammalian models are frequently used for preclinical evaluation of new drug delivery systems (DDS). However, valid mammalian models are expensive, time-consuming, and not easy to set up and evaluate. Furthermore, they are often linked to administrative burden with respect to ethical and legal aspects. The present review outlines the possibilities and limitations of using the hen's embryo, and specifically its chorioallantoic membrane (CAM), as an alternative to mammalian models for the evaluation of DDS. Features of the CAM, the anatomy of the embryo, and the blood were investigated to assess properties of the drug carriers such as toxicity and biocompatibility, as well as the activity, toxicity, biodistribution and pharmacokinetics of the drug. The simplicity, rapidity, and low cost of the different assays that can be performed with chick embryos strengthen the interest of routinely using this model in pharmaceutical technology research. It is concluded that there is a big potential for using chick embryos in screening procedures of formulation candidates, thus establishing an intermediate step between in vitro cellular tests and preclinical mammalian models. © 2007 Elsevier B.V. All rights reserved. Keywords: Chick chorioallantoic membrane (CAM) model; In vivo model; Preclinical evaluation; Angiogenesis; Drug activity; Cancer models; Photodynamic therapy; Biocompatibility; Pharmacokinetics; Biodistribution

Contents 1. 2. 3.

Introduction . . . . . . . . . . . . . . . . . . . . . Chick embryo and its chorioallantoic membrane . . Chick embryos for drug delivery systems evaluation 3.1. Chick embryo culture . . . . . . . . . . . . 3.2. Administration of the formulations . . . . . .

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Abbreviations: AβBA, acetyl-β-boswellic acid; AKαBA, acetyl-11-keto-α-boswellic acid; AKβBA, acetyl-11-keto-β-boswellic acid; 5-ALA, 5-aminolevulinic acid; AMD, age-related macular degeneration; BEPPn, bis(methoxyethyl)-di-n-propylporphycene; BMP-2, bone morphogenetic protein-2; BPD-MA, benzoporphyrin derivative monoacid ring A; CAM, chorioallantoic membrane; CD, γ-cyclodextrin; Ce6, chlorine e6; CNV, choroidal neovascularization; DDS, drug delivery system; DOX, doxorubicin; DPPC, dipalmitoylphosphatidylcholine; DT, diphtheria toxin; EDD, embryo development day; EPC, egg phosphatidylcholine; EVA, ethylenevinyl acetate; FDA, United States Food and Drug Administration; bFGF, basic fibroblast growth factor; HET-CAM, hen's egg test on the chorioallantoic membrane model; HPLC, high performance liquid chromatography; Hy, hypericin; IP, intraperitoneal; IV, intravenous; MB, methylene blue; MePEG, methoxypoly(ethylene glycol); NMP, N-methyl pyrrolidone; NP, nanoparticle; PCL, poly(ɛ-caprolactone); PDT, photodynamic therapy; PEG, poly(ethylene glycol); Pheo-a, pheophorbidea; PLA, poly(D,L-lactic acid); PLGA, poly(lactide-co-glycolide); PP IX, protoporphyrin IX; PS photosensitizer (s); PVP, polyvinylpyrrolidone; PVP-I, polyvinylpyrrolidone-iodine; SAIB, sucrose acetate isobutyrate; S1P, sphingosine 1-phosphate; TCPP, meso-tetra-(carboxyphenyl)porphyrin; TGF-β1, transforming growth factor beta-1; THPC, meso-tetra(m-hydroxyphenyl)chlorin; THPP, meso-tetra(p-hydroxyphenyl)porphyrin; TPP, meso-tetraphenylporphyrin; VEGF, vascular endothelial growth factor. ☆ This review is part of the Advanced Drug Delivery Reviews theme issue on “Prediction of Therapeutic and Drug Delivery Outcomes using Animal Models”. ⁎ Corresponding author. Tel.: +41 22 379 65 73; fax: +41 22 379 65 67. E-mail address: [email protected] (F. Delie). 0169-409X/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.addr.2007.04.019

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3.3.

Drug activity evaluation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3.1. Effects on the vasculature of the chorioallantoic membrane . . . . . . . . 3.3.2. Effects on tumors grown on the surface of the chorioallantoic membrane . 3.3.3. Effects on the development of the embryo . . . . . . . . . . . . . . . . . 3.4. Pharmacokinetics and biodistribution . . . . . . . . . . . . . . . . . . . . . . . . 3.4.1. Blood sampling and organ extraction . . . . . . . . . . . . . . . . . . . 3.4.2. Fluorescence measurements . . . . . . . . . . . . . . . . . . . . . . . . 3.4.3. Biosensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Advantages and limitations of chick embryos for evaluating drug delivery systems . . . . . . 5. Conclusions and perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction Nowadays, the pharmaceutical field faces an ever growing demand for making innovative formulations that are able to “intelligently” deliver modern active compounds. The development of high-throughput strategies for the discovery, synthesis, and screening of drugs, as well as the advances in genomics and proteomics, have resulted in a huge amount of new drug candidates for which formulations need to be designed. In this context, drugs, such as peptides, proteins, monoclonal antibodies, and nucleic acids, are particularly difficult to formulate. Progress in both polymer and biomaterial sciences offers the possibility of using new excipients for developing improved drug carriers. Ideally, delivery systems should have adequate drug loading, remain stable during storage and biodistribution, accumulate selectively at the target site, be able to release the drug in a controlled manner, be biodegradable, and perfectly biocompatible. To achieve these requirements, formulations have become increasingly complex. Although the use of high-throughput systems may accelerate both the formulation phases and the in vitro evaluation of drug delivery systems (DDS) [1], preclinical assays using mammalian models are still time-consuming. Furthermore, limits from ethical and legal points of view for working with those models are already very restrictive and are increasing steadily [2]. The chick embryo is a well-known animal model, which has been extensively studied from Aristotle's time—who opened hen's eggs daily to examine progressive stages of embryogenesis [3]—until the modern molecular era. The increasing interest in the chick embryo as a model in biological and pharmaceutical research is related to its simplicity and low cost compared with mammalian models. Current laws regulating animal experimentation in the USA, the European Union, and Switzerland allow experimentation with chick embryos without authorization from animal experimentation committees, on the grounds that experiments begin and end before hatching. Nevertheless, experimentation with chick embryos must be refined to reduce the number of embryos used through adequate experimental design. The present review outlines the potential use of the chick embryo, and specifically its chorioallantoic membrane (CAM), as an alternative to mammalian models for the evaluation of DDS. The CAM of the developing chick embryo is an extraembryonic membrane mediating gas and nutriment exchanges until hatching. Since the CAM has a very dense capillary network, it is commonly used to study in vivo both new vessel formation

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(angiogenesis) and its inhibition in response to different factors. In the literature, the use of CAM in research is referred to as the CAM assay or as the CAM model. Although chick embryos are not widely used for the evaluation of new drug carriers, the United States Food and Drug Administration (FDA) has approved products preclinically evaluated with chick embryos. Indeed, a FDA guidance for industry, published in June 2006, regarding the development of products for the treatment of chronic cutaneous ulcer and burn wounds, considered the CAM model as an alternative for preclinical testing. Various papers summarized the different applications of the CAM model in areas of interest for the pharmaceutical community, such as angiogenesis and antiangiogenesis [4–6], wound healing [7], tissue engineering [8], biomaterials and implants [9– 11], and biosensors [12]. The scope of the present review has been restricted to studies regarding the evaluation of DDS with the CAM model. The biological and physiological characteristics of the CAM are presented, as well as the protocols for embryo cultivation and administration of formulations. The use of chick embryos for the evaluation of drug activity, toxicity, biocompatibility, pharmacokinetics, and biodistribution are also summarized, concluding with an outline on the advantages and limitations, as well as some perspectives in the use of this model in pharmaceutical research. 2. Chick embryo and its chorioallantoic membrane Chick embryo development lasts 21 days before hatching. Hamburger and Hamilton classified the embryo development depending on a series of stages designed based on the external characteristics of the embryo [13]. The 21-day incubation period corresponds to 46 stages, known as the HH stages, which are not uniformly distributed over the time of development. However, in most studies on DDS, a different classification is followed: the first day of incubation is considered as the first day of embryonic development, termed as embryo development day (EDD). Three extraembryonic membranes protecting and nourishing the embryo are formed during development: the yolk sac membrane, the amnion, and the CAM. The latter is a transparent and highly vascularized membrane, formed during the EDD 4 to 5 by the fusion of the mesodermal layers of both the allantois and the chorion, resulting in a highly vascularized mesoderm composed of arteries, veins, and an intricate capillary plexus (Fig. 1) [5,14]. Rapid capillary proliferation continues until day 11. The mitotic

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Fig. 1. Chick embryo chorioallantoic membrane (CAM). a) CAM image taken from the top of the egg after opening the eggshell at day 10 of incubation. b) Localization of the CAM (in red) around the embryo and in direct contact with the eggshell at day 12 of incubation. c) Illustration of a cross-section of the CAM at day 10–12 of incubation. 1: chorionic epithelium; 2: mesoderm with blood vessels depicted in red; 3: allantoic epithelium; adapted from [14].

index then dramatically decreases and growth rate stays minimal [5,6,15]. The vascular system of the chick embryo attains its final arrangement on day 18, just before hatching [5]. The main function of the CAM is to serve as the respiratory organ for the embryo. The CAM also plays a role in the storage of excretions, electrolyte transport (sodium and chloride) from the allantoic sac, and mobilization of calcium from the shell to start bone mineralization. It is connected to the embryonic circulation by the allantoic arteries and veins, which are associated with lymphatic vessels [5]. Several features of the CAM change during embryo development, such as the composition of the extracellular matrix, the degree of differentiation of both endothelial cells and vessels, the characteristics of the interendothelial junctions, as well as the location of the vessels within the CAM [5]. This issue emphasizes the importance of always using embryos at the same EDD, failing which, comparisons between experiments can become problematic. As other vertebrates, chickens are protected by a dual immune system composed of B lymphocytes or B cells, and T lymphocytes or T cells, controlling the antibody—and the cell— mediated immunity, respectively. The B cells are differentiated in the bursa of Fabricius, the organ equivalent to the bone marrow in mammals, whereas T cells are differentiated in the thymus [16,17]. Until EDD 10, the chick embryo immune system is not completely developed. It lacks both B and T cell-mediated immune functions. Consequently, the young embryos are not fully immunocompetent. The presence of T cells can be first detected at EDD 11 and of B cells at EDD 12 [18]. By day 12, mononuclear phagocytes are found in the yolk sac, spleen, bursa, gut, thymus, and in the liver; and reticulum cells are present in the spleen and liver [18]. After EDD 15, the B cell repertory begins to diversify, and by EDD 18 chicken embryos become immunocompetent. Mammals have a wider spectrum of antibody specificities, with five forms of immunoglobulins: IgM, IgG, IgA, IgD, and IgE, whereas in chicken, there are only three forms: IgM, IgG, and IgA [17]. The ways in which the CAM and mammalian tissues respond to materials applied at their surface is important for evaluating topically applied DDS. Valdes et al. found that both acute and chronic inflammatory responses of the CAM to biomaterials are similar to those found in mammals [10].

3. Chick embryos for drug delivery systems evaluation Pharmaceutical technology scientists make huge efforts to produce new delivery systems capable of regulating the rate of drug delivery, sustaining the duration of the therapeutic action, and/or targeting the delivery of drugs. During the development of DDS, chick embryos can be used to evaluate the activity or toxicity of a drug on both the CAM and CAM-grafted tumors, as well as on the development of the body of the embryo. Toxicity of drugs or carriers on chick embryos can be evaluated in terms of embryo death or adverse effects on the CAM, including inflammation and neovascularization. The influence of the drug administration route can also be evaluated. Blood analysis, biosensors, or angiographies of CAM can be used to determine drug pharmacokinetic parameters. Different possibilities for the evaluation of DDS are depicted in Fig. 2. Often, the effects of DDS on the morphology of CAM are evaluated, although analyses of

Fig. 2. Use of the chick embryo for the evaluation of drug delivery systems (DDS). DDS can be topically applied on the chorioallantoic membrane or injected intravenously, intraperitoneally, into the yolk sac or into the amnion. After drug distribution within the embryo, drug activity, toxicity, biodistribution and pharmacokinetics (PK) can be determined. Biocompatibility of the DDS can be evaluated after placement on the CAM surface. When tumors are grafted on CAM, anticancer therapies can be evaluated.

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the blood and of the body of the embryo are also feasible. Table 1 lists DDS that have been evaluated with chick embryos. 3.1. Chick embryo culture The fertilized hen eggs are incubated at 37 °C under 60–70% relative humidity. Two approaches for embryo culture have been developed: “in ovo” method, where the embryos are left inside the eggshell during their development and for the duration of the assay, and “ex ovo” method, where the embryos are cultivated in recipients simulating the eggshell. The latter method is also known as shell-less culture. The choice of the method depends on the age of the embryo at the beginning of the experiment, the time the embryo is cultured, and the nature of the intervention. Table 2 lists the advantages and limitations of both methods. 3.2. Administration of the formulations Administration routes that can be used with chick embryos are depicted in Fig. 2. Similar to the administration routes used

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on humans, topical, intravenous (IV) [19–23], and intraperitoneal (IP) [24,25] administration can be used on chick embryos. Other routes with no equivalence in humans, such as injection into the yolk sac [26] or in the amnion [27], can also be used. For topical administration, the formulation is applied on the CAM surface. This route is the most commonly used one, owing to the accessibility to the CAM by simply opening the eggshell. Although IV administration is feasible, the difficulty to insert needles into the CAM vessels is a critical limitation. The volume of IV injection varies from 1 to 100 μl, depending on the EDD. The blood volume increases during the development of the chick embryo [28]; hence larger volumes can be injected at more advanced stages of embryo development. 3.3. Drug activity evaluation 3.3.1. Effects on the vasculature of the chorioallantoic membrane One important step before evaluating the efficiency of DDS, in terms of modification of the membrane, is the evaluation of

Table 1 Drug delivery systems (DDS) and formulations evaluated with chick embryos DDS

Drug a

Drug Type

Admin. route

Evaluated feature

Ref.

Drug-conjugates

AβBA, AK(αβ)BA Ce6

AA PS

Efficacy, PK Uptake, PK

[70,81] [72]

DOX DT Amiloride S1P BPD-MA

AA AA AA PA PS

Efficacy, ET Efficacy Efficacy Efficacy Uptake, efficacy

[55] [56] [54] [46] [24,25,97]

BPD-MA BPD-MA DOX MB Porphycenes

PS PS AA PS PS

Uptake, efficacy Efficacy, PK Efficacy Uptake, efficacy Efficacy, PK

[24] [19] [71] [74] [26,65]

PP IX PVP-I THPC Fluorescent dye No drug Paclitaxel Ce6, TCPP, TPP, Pheo-a THPP L-thyroxine Paclitaxel Calcitrol No drug 5-ALA and derivatives BMP-2 bFGF, VEGF bFGF, TGF-β1 VEGF VEGF VEGF VEGF Hypericin

PS AM PS – – AA PS PS H AA H – PS PA PA PA PA PA PA PA PS

Topic Topic Systemic Topic Topic Topic Topic Topic Systemic Systemic Systemic Topic Topic Topic Systemic Systemic Topic Systemic Systemic Topic Topic Systemic Systemic Topic Topic Topic Topic Systemic Topic Topic Topic Topic Topic Topic Topic Topic

ET Biocompatibility Efficacy, PK Extravasation Biocompatibility Efficacy Efficacy, PK Efficacy, PK Efficacy, ET Efficacy Efficacy Biocompatibility Biodistribution Efficacy Efficacy Efficacy Efficacy Efficacy Efficacy Efficacy Uptake, efficacy

[27] [33] [22] [53] [32] [49,50] [21,23] [20] [76] [51,52] [77,78] [29] [82] [48] [40] [45] [41] [42] [43] [44] [62–64,73]

Gels Liposomes

Lipospheres Microemulsions Microparticles Nanoparticles Osmotic pumps Pastes Pellets Prodrugs Scaffolds

Solutions

a For drug abbreviation see the abbreviation list, AA: antiangiogenic, AM: antimicrobial, ET: embryotoxicity, H: hormone, PA: proangiogenic, PK: pharmacokinetics and PS: photosensitizer.

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Table 2 Characteristics of the “in ovo” and “ex ovo” chick embryo culture [4,6,38] In ovo Description Embryos are left inside the eggshell during their development and for the duration of the assay. Advantages –The source of calcium for building skeletal elements is kept. –Normal development of the embryo. –High embryo survival rate. –Easy methodology. –Sterility is not required. –Embryos can reach hatching. Limitations –Small surface is exposed. –Difficult monitoring. –Risk of angiogenesis induced by eggshell pieces.

Ex ovo (shell-less) After opening the eggshell, the embryo and its extraembryonic membranes are transferred to Petri or plastic dishes and further incubated outside the eggshell. –Large CAM area available for testing. –Direct visualization of the entire CAM. –Evaluation of several samples in one single embryo. –Easy grafting and monitoring of excised tissues. –No eggshell falling on CAM. –Easy access to CAM vasculature. –Possibility of transillumination. –In vivo observation of embryo development. –Difficult methodology (e.g. transfer the embryo to plastic dish, sterility requirements). –Low embryo survival rate. –Do not reflect physiological conditions. –Embryos cannot reach hatching.

the biocompatibility and toxicity of both the carrier and the drug. The biocompatibility of materials used for DDS production is usually evaluated in vivo using mammalian models, such as mice, rats, and dogs, after implantation of the material. Implantation in mammalian models does not allow continuous evaluation of the tissue reactions to the implant, but rather requires animal sacrifice, tissue sampling, processing, and histological evaluation [9,10]. Zwadlo-Klarwasser et al. and Valdes et al. demonstrated that the CAM model can be used as an alternative for studying the angiogenic and inflammatory response to biomaterials [9,10]. The CAM model allows a continuous visualization of the implant site and provides a rapid, simple, and low cost screening of tissue reactions to biomaterials [9,10]. Indeed, the chemical composition of the carrier, as well as its structure (e.g. porosity, smoothness, geometry), influenced the inflammatory or angiogenic responses of CAM [9,10]. The biocompatibility of pellets made of ethylene-vinyl acetate (EVA) copolymer and bovine serum albumin were evaluated after topical application on the CAM [29]. No signs of inflammation, edema or neovascularization were observed, demonstrating the pellet's biocompatibility. Similarly, the field of cosmetics has used the CAM model to evaluate the irritation properties of cosmetic formulations and ingredients [30]. This assay, namely HET-CAM (hen's egg test on the CAM model), was proposed as an alternative to the Draize rabbit eye irritation test [31]. In the field of microemulsions for ocular delivery, with chick embryos, the irritation potential was shown to be independent of the microemulsion microstructure, but dependent on the irritative properties of the excipients [32]. The irritative potential of drugs incorporated in DDS can also be evaluated with the CAM model. Polyvinylpyrrolidone-iodine (PVP-I), an antimicrobial compound used as a topical disinfectant, was encapsulated in phosphatidylcholine liposomes to reduce irritation from conventional preparations such as solutions [33]. Topical application on the CAM demonstrated that PVP-I was better tolerated in the liposomal than in the aqueous formulation. However, the influence of PVP-I incorporation into liposomes on the antimicrobial activity was not evaluated in this study. Once the drug and the carrier toxicity have been verified, drug efficacy may be determined. Many drugs evaluated with the CAM

model induce modifications of the vasculature. In the case of proangiogenic drugs, an increase of the number of vessels is observed (spoke-wheel formation), whereas antiangiogenic drugs or photodynamic treatment induce vascular occlusion or cause decrease in the number of vessels. The evaluation of these treatments varies from a simple qualitative observation, such as the assessment of hemorrhages, vascular lysis, and coagulation [29,33], to sophisticated evaluations of CAM vessels after computerized treatment of images, including fractal analysis [34], and infrared tomographic imaging [35]. Strategies to examine drug effects on CAM vasculature have been extensively reviewed by Richardson and Singh [6]. Quantification can be made by counting the intercepts of vessels with one or a series of concentric circles. Score systems are mainly based on the number, diameter, length, branching degree, and order of new or occluded vessels in a specific area of the CAM [6]. 3.3.1.1. Proangiogenic drugs. Angiogenesis, the physiological process involving the growth of new blood vessels from preexisting vessels, is essential for organ growth and repair. However, the imbalance in angiogenesis contributes to numerous pathologies, such as cancer, age-related macular degeneration, rheumatoid arthritis, and inflammation [36]. On the other hand, angiogenesis can be advantageously used for wound healing, and for increasing the compatibility, functionality, and integration of any implanted medical device [37]. Several models for in vivo evaluation of angiogenesis and antiangiogenesis exist and have been already reviewed [38,39]. Chamber techniques, such as the dorsal skinfold chamber, are used to visually monitor the progress of neovascularization induced by implanted tumors or proangiogenic substances in mammalian models. The corneal angiogenesis assay in rabbits, rats, and mice is based on the stimulation of vessel growth in the cornea, an avascular tissue. The subcutaneous implantation of materials, such as Matrigel™, containing cells or substances inducing angiogenesis has also been used for angiogenesis evaluation [38,39]. Several days after implantation, the implant is extracted from the animal and examined for vessel growth. The CAM model offers advantages that include the comparative ease of culture, low cost, and easy observation of the neovascularization while the embryo is

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still alive, without the need to sacrifice the animals at each timepoint. The CAM model allows one to evaluate the extent of angiogenesis, as well as the quality of the newly formed vessels, when proangiogenic growth factors, such as the vascular endothelial growth factor (VEGF) or the basic fibroblast growth factor (bFGF), are administered for stimulating neovascularization. Fibrin matrices containing bFGF, VEGF121, or VEGF165, displayed angiogenic activity in the CAM model, but the newly formed capillaries were leaky and hemorrhages within the fibrin matrices were observed [40]. On the contrary, no hemorrhages occurred when the factors were delivered in combinations: VEGF121 plus VEGF165, or VEGF165 plus bFGF [40]. The authors thus suggested that the new vessels were more mature than those produced by single factors. In contrast, Ehrbar et al. demonstrated with the CAM model that VEGF alone can initiate the formation of structurally intact vessels when it is released slowly in a low and sustained dose [41]. To avoid the burst release of VEGF from fibrin matrices, a variant of VEGF (α2PI1–8-VEGF121) was covalently linked to the fibrin network (Fig. 3). Since the α2PI1– 8 domain is cleaved via enzymatic activity, VEGF is only released from the fibrin matrix as a consequence of the activity of cells that locally remodel the fibrin matrix. The VEGF121 in fibrin matrices induced the formation of malformed and leaky vessels with chaotic perturbations and disturbance of the hierarchy. In contrast, when α2PI1–8-VEGF121 was covalently linked to the fibrin matrix, vessel growth was more controlled and closer to normal architecture, as demonstrated by the histology of CAM crosssections, and 3D images obtained after corrosion casting of CAM vasculature [41]. This study revealed the critical role of the release rate of VEGF. Normal vasculature growth was observed when α2PI1–8-VEGF121 was slowly released from the matrix after enzymatic cleavage, whereas the diffusive burst release of VEGF121 induced aberrant angiogenesis. Studies of vessel permeability in mice further validated the results. Biocompatible collagen matrices were identified as tissue substitutes or scaffolds for tissue regeneration. Collagen matrices were modified by covalent linkage with heparin to increase the ability of collagen to support the proliferation of endothelial cells [42]. The number of capillaries (diameter smaller than 20–40 μm) was higher for the embryos receiving the collagen matrices linked to heparin than for those receiving unmodified matrices. Further experiments demonstrated that the angiogenic potential of heparin-

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collagen matrices increased by the incorporation of VEGF165. Another study showed that the covalent attachment of VEGF165 to collagen matrices resulted in higher angiogenic activity than simple VEGF165 incorporation into the collagen matrix [43]. In vitro release tests suggested that the slower release of VEGF165 when linked to the collagen matrix was responsible for the higher activity of this formulation. Unfortunately, the quality of the neovasculature was not evaluated in these studies, and, as outlined before, the stimulation of angiogenesis can result in the formation of vessels of different morphology. Poly(D,L-lactic acid) (PLA) was used to produce porous biodegradable scaffolds incorporating VEGF165 [44]. Four days after placement of the scaffold on the CAM of embryos at EDD 10, vessel formation around the scaffold was observed demonstrating that VEGF165 incorporated into PLA scaffolds was released in an active form stimulating angiogenesis. Acellular matrices, which are the noncellular part of a tissue, have been used in tissue regeneration studies. They can be transplanted without rejection and provide a matrix where cell growth, angiogenesis, and differentiation can occur. The angiogenic activity of acellular femoral matrices, obtained from rat femurs, was evaluated with embryos cultivated “in ovo” [45]. A strong angiogenic response (spoke-wheel pattern) was observed 4 days after matrix application. The incorporation of bFGF and transforming growth factor beta-1 (TGF-β1), angiogenic cytokines involved in bone formation, further increased the angiogenic response of the acellular matrices. Initially, modulation of angiogenesis for therapeutic purposes was intended to induce new vessels formation. However, recent studies revealed the importance of using factors capable of providing a better stabilization of the new vasculature. Indeed, longer-term clinical studies have showed that newly formed vessels may be unstable, leading to disintegration and minimal long-term improvement of function [46]. Sphingosine 1-phosphate (S1P), a factor released from activated platelets and capable of controlling angiogenesis, was incorporated into hydrogels made of poly(ethylene glycol) (PEG) [46]. PEG-octavinyl sulfone was crosslinked with either albumin or PEG-diamine. Hydrogels crosslinked with albumin induced a moderate to strong angiogenic response in the CAM model, whereas crosslinking with PEG-diamine resulted in a modest angiogenic response. This study demonstrates that the interaction of albumin immobilized within the gel is necessary to control S1P release. Indeed, S1P is

Fig. 3. Cell regulated release of VEGF from fibrin matrices. a) VEGF release by diffusion. b) When a covalent linkage is formed between α2PI1–8-VEGF121, a recombinant variant of VEGF, and the fibrin matrix, VEGF passive diffusion is avoided. VEGF121 is released by cleavage from the fibrin matrix only via cellassociated enzymatic activity represented by the arrow. Adapted from [41].

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transported in the blood by proteins, such as albumin and highdensity lipoproteins. Angiogenesis is essential in providing necessary blood supply for longitudinal bone growth and fracture repair [47]. The formation of cartilage and bone for orthopedic applications was evaluated with an ex vivo model of bone formation developed on the CAM model [48]. Chick embryos were incubated until EDD 18, when chick femurs were excised. Then, a segmental defect was created in the femur, into which a PLA scaffold, containing both bone morphogenetic protein-2 (BMP-2) and human bone marrow seeded cells, was placed to fill the defect site. Then, the chick-excised femurs containing the scaffolds were placed on the CAM of other embryos at EDD 10. The explants were harvested at EDD 17 for histochemical analysis. Extensive angiogenesis, and new cartilage and bone formation were observed within the chick bone defect. However, controls of scaffolds without BMP-2 or cells were not evaluated. This study demonstrated that the traditional CAM assay could become a valuable model for bone regeneration studies, owing to the potential for CAM vessels to invade any tissue explants implanted on it. 3.3.1.2. Antiangiogenic drugs. Abnormal angiogenesis has a critical role in numerous malignant, inflammatory, ischemic, infectious, and immune disorders [36]. Since tumor growth is dependent on angiogenesis, the goal of cancer therapy with antiangiogenic drugs is the occlusion of the blood vessels feeding the tumor. The efficiency of formulations containing antiangiogenic drugs with the CAM model may be easily evaluated by the assessment of the vessel occlusion. Several formulations of paclitaxel, a hydrophobic anticancer drug with antiangiogenic properties, were evaluated with the CAM model and are summarized in Table 3. Paclitaxel was loaded in microspheres of either poly(ɛ-caprolactone) (PCL) [49] or a blend of PLA and EVA copolymer [50]. When microspheres made of PLA-EVA were used, vascular occlusion was achieved with a paclitaxel dose approximately eight times smaller than the dose used for PCL microspheres. Although, in vitro paclitaxel release was higher for microspheres made of PCL, PLA-EVA microspheres were more active in vivo. These studies demonstrated that the CAM model allowed one to compare the performance of different polymers for producing microspheres. Injectable polymeric surgical pastes were investigated for localized paclitaxel delivery, to act as a cancer prophylactic agent during or immediately after cancer resection. Paclitaxel was incorporated into blends of PCL and methoxypoly(ethylene

glycol) (MePEG), used for decreasing the melting point for injection [51]. Pastes containing 0.25% paclitaxel and higher loadings induced the formation of avascular zones. Both MePEG and paclitaxel decreased the melting point of the PCL matrix, but a decrease in the release of paclitaxel rate was observed [51]. A strategy to increase paclitaxel release rate was further developed and evaluated with the CAM model by replacing the MePEG in the PCL matrix by coprecipitated microparticles of paclitaxel with gelatine, a water-soluble polymer [52]. As a proof of the concept, the ultrasound-triggered release of a fluorescent dye incorporated in lipospheres, intended for paclitaxel delivery, was evaluated with the CAM model [53]. Lipospheres, composed of a lipid shell conjugated to a targeting peptide (specifically binding αVβ3 integrins), an oil incorporating a fluorescent dye, and a gas core that allows acoustical manipulation, were IV administered to chick embryos. One minute after topical ultrasound application, fluorescence was observed at both the endothelium and the extravascular space, whereas no extravascular fluorescence was observed in the absence of ultrasound. The studies dealing with paclitaxel [49–52] used a semiquantitative evaluation with only two possible responses: positive or negative angiogenic activity, depending on the appearance of an avascular zone of a determined diameter (Table 3). However, this kind of evaluation is very limited due to intermediate responses, such as the occlusion of very small capillaries, which are not taken into account. Furthermore, a large zone of avascularity can indicate both a high diffusion of the drug from the site of application, as well as a high effect on vascular inhibition. Quantitative methods for evaluating the changes of CAM vasculature have been developed such as fractal analysis [34]. Fractal dimension was used to determine the extent in inhibition of angiogenesis induced by amiloride released from two polymerbased gels [54]. At EDD 7 and 8, gels of either sucrose acetate isobutyrate (SAIB) or calcium alginate containing amiloride were applied on the CAM. Fractal dimension values increased in control embryos, representing greater vascular complexity or increased branching. On the contrary, fractal dimension values decreased in embryos receiving the gels with amiloride. Although, the in vitro release of amiloride in water was higher from calcium alginate than from SAIB gels, these vehicles were equally effective in inhibiting angiogenesis in the CAM model. Doxorubicin (DOX), an anticancer compound with antiangiogenic properties, was conjugated to PEG of 20 kDa in order to increase water solubility and half-life circulation [55]. The PEGDOX conjugate contained an acid-sensitive hydrazone group,

Table 3 Evaluation of paclitaxel formulations with the CAM model Delivery system

Paclitaxel dose

Observation

Ref.

Microspheres made of PCL

50 μg

[49]

Microspheres made of a blend of PLA and EVA copolymer Paste (blend of polymers) made of PCL and Me(PEG)

6 μg 1.5–600 μg

Paste of PCL containing microparticles of gelatine

20 μg

Avascular zone of 2-6 mm in diameter for loaded microspheres. Normal vasculature when unloaded microspheres were used. Idem. Paclitaxel doses of 7.5 μg and higher induced avascular zones measuring at least 4 mm in diameter. Unloaded pastes did not alter the vasculature. Avascular zone of 2-6 mm in diameter for loaded paste. Normal vasculature when unloaded paste was used.

The response was evaluated 2 days after topical application on embryos at EDD 6 cultured “ex ovo”.

[50] [51] [52]

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which cleaves in the acidic pH of tumors, thus releasing the drug. Using the CAM model, the PEG-DOX conjugate was shown to be less embryotoxic than DOX. However, conjugation to PEG resulted in the complete loss of DOX antiangiogenic activity. These results suggest that this prodrug was inefficient per se and that the lack of activity was most likely because CAM does not have an acidic environment to cleave the conjugate and release the drug. The VEGF can be used to target drugs to the tumor vasculature that overexpresses the VEGF receptor [56]. Recombinant VEGF was linked to a truncated form of diphtheria toxin (DT) lacking the receptor-binding domain [56]. Once coupled to VEGF, the cytotoxicity of DT is expected to occur specifically in endothelial cells. In this study, the effects of the conjugate were evaluated both on vessels preestablished on CAM and on recently formed neovessels, that were induced by the application of bFGF, another angiogenic compound. The neovascularization induced by bFGF, was totally blocked by the cotreatment with the VEGF-DT conjugate, whereas the preestablished vessels were not affected by the conjugate. These experiments demonstrated that a toxin conjugated to VEGF is able to block growth factor-induced vascularization in vivo. Free toxin or a mixture of toxin and VEGF did not inhibit the bFGF-induced neovascularization, indicating that VEGF and DT must be chemically conjugated for efficient targeting. Unconjugated DT was inactive because it lacked the receptor-binding domain. Investigation with DDS incorporating pro-and antiangiogenic drugs demonstrated that the CAM model allows one to evaluate drug activity as function of: (i) the formulation type, (ii) the excipients used for producing DDS, and (iii) the drug release rate. The utility of using drug combinations against single drugs, and drug conjugation for solubilization or active targeting, can be evaluated with the CAM model as well. 3.3.1.3. Photosensitizers. Although the activity of photosensitizers (PS) results in vessel occlusion, as is the case of some antiangiogenic compounds, PS are described separately because they are not active per se, but rather need to be activated by light. Photodynamic therapy (PDT) is based on PS administration and further illumination of the target site, for activating the PS in the presence of oxygen. Photochemical reactions are thus generated triggering biochemical, immunological, and physiological cascades, finally resulting in the destruction of the irradiated tissue [57]. The main therapeutic applications of PDT are cancer therapy [58] and the treatment of neovascularization-related disorders,

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such as choroidal neovascularization (CNV) secondary to agerelated macular degeneration (AMD) [59]. In cancer treatment, PDT induces the death of tumoral cells and the occlusion of the blood vessels feeding the tumor, whereas in CNV-AMD treatment, the main goal is to occlude the pathological neovascularization that originates the disease. For cancer applications, PDT is frequently evaluated in mammalian models bearing tumors. In the case of CNV-AMD treatment, rodents in which CNV has been triggered by either laser injury or external factors, such as adenovirus [60], as well as genetically modified animals developing CNV [61], are used. The chick embryo can be a suitable tool to screen the effects of different pharmaceutical formulations of PS, particularly on the photo-induced vascular occlusion of CAM, and on the regression of tumors grafted on the CAM surface. The PDT on chick embryos implies different steps. The PS is administered topically or systemically to the embryo, often by IP or IV injection. After a defined postadministration time, allowing for the absorption and biodistribution of the PS, PDT is performed by irradiating the CAM with a laser or filtered light. The PDT induces vascular occlusion, which is easily visualized by microscopy (Fig. 4). Because of its simplicity, topical application is often used with the CAM model, but is of interest only for studies related to surface-accessible lesions or dermatological conditions. Systemic administration by IP injection is closer to the administration route used in mammalian models and clinical practice. The feasibility of using this administration route was evaluated with liposomal benzoporphyrin derivative monoacid ring A (BPDMA) and other PS. The IP administration resulted in higher vascular damages when compared with topical application [25]. Since topical or IP application of PS is not relevant for the CNV-AMD treatment, IV injection was used by Lange et al. for administering Visudyne® (BPD-MA encapsulated in liposomes), the first approved PS for clinical PDT of classic subfoveal CNV, to embryos at EDD 12 [19]. The CAM model exhibited a similar response to that obtained in clinics. Moreover, the effects of the long-term storage of liposomal BPD-MA solutions were tested. A solution reconstituted 2 weeks before use and stored at 4 °C, induced no vascular occlusion in the CAM. The reduced phototoxicity may be due to either the instability of liposomes or the degradation of the PS itself. As most PS used for PDT, Hypericin (Hy) is hydrophobic. To solve the problem of administration, a Hy formulation was developed containing a biocompatible solvent, the N-methyl

Fig. 4. Typical photodynamic therapy experiment observed by fluorescence angiography. a) After the administration of PS to chick embryos, the circulating PS can be observed by using appropriate excitation and emission filters; b) PDT is performed by irradiating CAM vessels with light. Illumination activates the PS, thus triggering the photochemical cascades responsible of the photodynamic effect; c) Twenty-four hours after treatment, injection of rhodamine, a fluorescent compound, allows the visualization of CAM vasculature and the assessment of the vascular occlusion induced by PDT.

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pyrrolidone (NMP), and 0.9% sodium chloride [62–64]. The NMP concentrations between 0.6% and 4.8% were well tolerated by embryos after topical application [64]. The Hy formulations containing either 0.6% or 4.8% of NMP were evaluated in terms of the antivascular effects on CAM vasculature induced after light irradiation [63]. The PDT response, evaluated by the vessel regression percentage, was NMP concentration-dependent. When 4.8% NMP was used, a twofold efficacy was observed. In this case, the use of NMP as a solvent seemed to enhance the permeability of the CAM to Hy and thus resulted in a higher photoactivity. Polymeric nanoparticles (NP) have been developed for IV administration of hydrophobic PS. Meso-tetra(p-hydroxyphenyl)porphyrin (THPP) was incorporated into NP made of poly (lactide-co-glycolide) (PLGA) [20]. The vascular damages induced by THPP entrapped into NP were compared with the PS formulated in solution. The incorporation of THPP in NP significantly enhanced its phototoxic effect on blood vessels. As a higher PDT effect was obtained with NP, drug dose can be reduced to achieve the desired therapeutic effects with reducing side effects. PLA NP were used by Pegaz et al. to deliver four different PS: meso-tetraphenylporphyrin (TPP), meso-tetra-(carboxyphenyl)porphyrin (TCPP), pheophorbide-a (Pheo-a), and chlorine e6 (Ce6) [21]. These PS differ in their hydrophilic/lipophilic balance, which strongly affects their incorporation in NP (drug loading: 0.5 and 4.6% w/w for Ce6 and TPP, respectively). The most lipophilic PS (TPP) led to the highest extent of vascular occlusion. The authors proposed that nanoencapsulation may improve the activity of PS by enhancing the PS uptake via NP endocytosis. With the CAM model, the NP size was shown as an important parameter for controlling the extent of the vascular occlusion. The activity of TCPP, incorporated in PLA NP increased with the decrease in NP diameter to around 100 nm [23]. The studies made on the CAM model showed that the type of carrier is crucial for the delivery and efficiency of the PS, but the PS properties, such as hydro/lipophilicity, polarity, etc., also influence its activity. Gottfried et al. evaluated the efficacy of five porphycene-type sensitizers encapsulated into liposomes [26]. Solutions of porphycenes in 10% dimethyl sulfoxide or dimethyl formamide were found to be toxic for the embryos. Thus, egg phosphatidylcholine (EPC) and dipalmitoylphosphatidylcholine (DPPC) liposomes were developed. At EDD 10, porphycene liposomal formulations were topically applied on the membrane. The liposome-entrapped porphycenes exhibited similar uptake kinetics as that of water-soluble PS. After PDT, liposomeencapsulated drug induced an increase in damage to blood vessels, as the number of polar substituents of the porphycene molecule increased. The PDT results thus were independent of the nature of liposomes (EPC or DPPC) used for PS delivery. Toledano et al. studied four porphycene derivatives after encapsulation into DPPC liposomes [65]. Spectroscopic measurements demonstrated that all PS were located in the phospholipid bilayer of the liposome, but their proximity to the membrane surface was dependent on their hydrophilicity. Porphycene

location in liposomes was consistent with in vitro activity evaluated on MDCK epithelial cells: the more the porphycene derivatives were closer to the liposomal surface, the more they were cytotoxic. However, in vivo, this parameter did not influence the CAM blood vessel injury. Indeed, efficient damages were observed even for hydrophobic PS located deeper in liposomes. The drug delivery occurred by different pathways in vitro and in vivo. Thus, results obtained on cell culture may not always be correlated to in vivo conditions, where lipoproteins present in the serum can increase the drug release. The CAM model offers the possibility to perform simple and cheap in vivo assays and thus do a more relevant formulation screening than in vitro tests. To enhance circulation time after IVadministration, liposomes can be coated with hydrophilic polymers such as PEG. Longer circulation times increase the probability to reach the target site [66]. Pegaz et al. compared the vascular occlusion induced by meso-tetra(m-hydroxyphenyl)chlorin (THPC) entrapped either into plain or PEGylated liposomes made of DPPC [22]. Liposome PEGylation improved the THPC photoactivity. Indeed, when using plain liposomes a twofold higher light dose was necessary to equal the damage induced by PEGylated liposomes. This effect is presumably due to the long-circulating properties of PEGylated liposomes, that have also been observed in mammals [66]. The CAM model offers several advantages for the evaluation of DDS incorporating PS. The well-vascularized membrane is easily accessible and easy to handle for administration of PS and irradiation. Because of the transparency of its superficial layers, nearly any wavelength in the visible part of the spectrum can be used for photodynamic treatments, fluorescence analyses of administered PS, and optical examination of PDT-induced vascular damage. Furthermore, photodynamic effects can be monitored in real-time, in individual blood vessels. 3.3.2. Effects on tumors grown on the surface of the chorioallantoic membrane Since the developing embryo is not fully immunocompetent, the CAM appeared to be an excellent host for cancer cells and biopsies from patients. When tumors are grown in this model, many in vivo characteristics of tumors, such as mass development, angiogenesis, infiltrative growth and metastasis, have been observed [67,68]. Indeed, tumor-induced angiogenesis has been studied with the CAM model since 1913, as described by Ribatti et al. [69]. Compared with mammalian models, where tumor growth often takes between 3 and 6 weeks, assays using chick embryos are faster. Between 2 and 5 days after tumor cell inoculation, the 3D tumor xenografts become visible and are supplied with vessels of CAM origin. Furthermore, several different tumors can be grown on the same membrane, allowing the comparison of positive and negative controls in the same embryo. Hydrophobic derivatives of boswellic acid, cytotoxic compounds extracted from Boswellia serrata, were conjugated with γ-cyclodextrin (CD) to increase water solubility [70]. The antitumor activity of these conjugates was then evaluated with chemoresistant PC-3 human prostate cancer cells grown on the CAM. Acetyl-11-keto-β-boswellic acid CD (AKβBA-CD) was about six-times more potent than acetyl-β-boswellic acid CD (AβBA-CD) in inhibiting tumor growth. Although small

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differences between AKβBA-CD and AβBA-CD were observed during in vitro assays on PC-3 tumor cells, the CAM model demonstrated that AKβBA-CD was superior [70]. The therapeutic efficacy of cancer active-targeting using DOX-loaded immunoliposomes was evaluated with the CAM model [71]. The DOX-loaded liposomes were coupled either to monoclonal antibodies targeting tumor cells (anti-GD2), or to NGR peptides that target tumor vessels. The antiangiogenic effects of these formulations were tested on xenografts derived from neuroblastoma cell lines grown on the CAM surface. When anti-GD2 or NGR liposomes were administered separately, 50– 60% of vessel growth inhibition was achieved, whereas administering a combination of both types of liposomes increased vessel growth inhibition to 90%. The higher efficiency of the combined treatment was further validated in tumor-bearing mice. Despite the ease of observation of both tumors and their vascularization on the CAM, few studies have been carried out on fluorescence photodetection and PDT of tumors. Chin et al. evaluated the possibility of using a hydrophilic derivative of the photosensitizer Ce6 for the fluorescence photodetection of bladder cancer [72]. Ce6 conjugated to polyvinylpyrrolidone (PVP) was tested on MHG bladder microtumors grown on CAM surface. Fluorescence imaging was performed 30 min after topical application of Ce6-PVP. Higher fluorescence was observed in the bladder tumor cells when compared with normal CAM tissue. Using the same model, Hy formulations containing either NMP or albumin, as solubilizing agents, were tested for the photodiagnosis of bladder cancer [73]. Topical application of Hy dissolved in 0.05% NMP, where Hy was aggregated, resulted in higher ratios between tumor and adjacent tissues than Hy in 0.5% albumin, where Hy was mostly monomeric. Although it has been suggested that aggregates cannot easily cross the cellular membranes, the authors proposed that the higher efficiency of the Hy-NMP formulation was due to an increased tumoral-cell uptake of aggregates. The tumor growth inhibition induced by bis(methoxyethyl)di-n-propylporphycene (BEPPn) incorporated into EPC and DPPC liposomes was evaluated in tumors, induced by the implantation of SV40-transformed bulb/3T3 fibroblasts between the ectodermal and mesodermal CAM layers [26]. Liposomes were systematically administered to the yolk sac and, after 24 h, tumors were irradiated with light. In these experiments, nonirradiated tumors grown on the same CAM continued to proliferate, while light-treated tumors showed typical signs of necrosis induced by PDT, independent of the nature of the lipids used for PS encapsulation. However, no comparison was made with a solution of BEPPn. Liposomes have been used also to avoid PS adverse effects on healthy tissues. For this purpose, fresh biopsies obtained from primary human malignant ovarian adenocarcinoma were implanted on CAM until satisfactory vascularization [74]. Then, the PDT effect after administration of methylene blue (MB), in solution or incorporated in soy lecithin liposomes, on the ovarian tumor was evaluated. In the first 2 days, the treated tumors were markedly decreased in size. Five days post-PDT, total remission of surviving embryos was obtained for both formulations. However, the aqueous MB solution induced more

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damage to healthy tissue, leading to higher embryo death, indicating that the liposomal formulation increases the tumortargeted delivery, and thus minimizes side effects. In conclusion, grafted tumors on the CAM surface have similar characteristics as the tumors grown in mammalian models, with the additional advantage that the setup of the CAM for cancer studies is faster. However, owing to the normal development of the chick embryo, long-term evaluations are not feasible. 3.3.3. Effects on the development of the embryo Drugs topically applied to the CAM can reach the systemic circulation, after absorption through the membrane, and affect the body development of the chick embryo. This modality has been particularly used for evaluating the activity of hormones and the embryotoxicity of compounds. The activity of hormones is usually studied in vivo in normal and genetically modified mammalian models. Embryotoxicity studies are made in pregnant animals, predominantly rats and rabbits, and in organ and whole embryo cultures [75]. The long-term topical delivery of thyroxine using osmotic minipumps was compared with systemic administration [76]. While long-term delivery resulted in 81% embryo survival, 50% of embryos died after single bolus injection. Extensive cell sloughing of the developing chick corneal epithelium appeared earlier when minipumps were used, demonstrating that slow delivery decreased embryo mortality and resulted in higher hormonal activity. Furthermore, the effects of calcitrol (1,25-dihydroxycholecalciferol) on the concentration of inorganic ions in the body of the embryo were evaluated after topical application of the hormone when incorporated in pellets made of cholesterol, methylcellulose and α-lactose [77,78]. Although these studies did not detail the pellet formulation, and no controls were made with the hormone administered in solution, they provided evidence of the potential use of chick embryos for evaluating hormone carriers. Nevertheless, for hormone evaluation the chick embryos must be further characterized to detect possible differences between avian and mammalian hormonal systems. The chick embryo has been used also for embryotoxicity studies [79,80]. Embryotoxicity, in terms of lethality and teratogenicity, of protoporphyrin IX (PP IX) was studied on embryos at EDD 4 after intraamniotic administration [27]. The toxicity in the presence and in the absence of light, namely light and dark embryotoxicity, respectively, were evaluated separately. Administration of PP IX in solution or incorporated in liposomes followed by light irradiation induced embryotoxic effects. Dark embryotoxicity was higher for PP IX incorporated in liposomes, probably due to PP IX enhanced penetration into embryonic tissues when administered in liposomes. Studying embryotoxicity on chick embryos is much simpler and faster to set up than investigations in pregnant mammalians. However, the main limitations are the interspecies differences in embryo metabolism between avian and mammalian models. 3.4. Pharmacokinetics and biodistribution One of the main objectives for developing DDS is the control of the drug release rate. Therefore, in vivo data on pharmacokinetics

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and biodistribution provide crucial information on the efficacy of DDS. Different approaches to assess pharmacokinetics and biodistribution have been used with chick embryos ranging from classical blood sampling and organ extraction followed by analytical quantification of the drug, to the use of biosensors or direct observation of intravascular fluorescence after administration of fluorescent drugs. 3.4.1. Blood sampling and organ extraction The pharmacokinetics of a hydrophilic derivative of boswellic acid (acetyl-11-keto-α-boswellic acid CD or AKαBA-CD) were evaluated in chick embryos after topical administration [81]. Plasma levels of the drug were determined by high performance liquid chromatography (HPLC) after blood sampling, and pharmacokinetic parameters were determined. This study demonstrated that the hydrophilic derivative of AKαBA topically applied on CAM reached the systemic circulation. Unfortunately, the hydrophobic counterpart was not evaluated in these studies. Pharmacokinetics was also evaluated on PC-3 tumor-bearing embryos [70]. Topical application of a solution of AKβBA-CD on the tumor yielded only low systemic plasma levels in the chicken embryo. Furthermore, no signs of toxicity were detectable during autopsy of embryos demonstrating that after topical application on the tumors, there was no systemic activity of the drug. Drug biodistribution can be assessed by quantifying drug concentration in different organs. Fotinos et al. demonstrated with the prodrugs 5-aminolevulinic acid (5-ALA) and two 1,3diacylglyceride 5-ALA derivatives that drug biodistribution can be assessed after dissection of different organs and CAM of embryos at EDD 11, followed by HPLC analyses [82]. Because of the low blood volume [28] and small organ size of chick embryos, very sensitive analytical methods must be used. Furthermore, the use of embryos strictly at the same timepoint after fertilization is imperative because the pharmacokinetic data change with the embryonic development [81]. 3.4.2. Fluorescence measurements In the case of fluorescent drugs, fluorescence measurements can be used to study drug biodistribution or pharmacokinetics after administration. The choice of the devices used for fluorescence detection should consider the optimal excitation and emission bands of the fluorescent drug, and the autofluorescence of CAM tissues. Two approaches were used: (i) tissue sampling and fluorescence analyses of tissue sections [74], and (ii) direct fluorescence measurements of CAM vasculature without resection [19]. Fluorescence microscopy was used to evaluate the distribution and penetration of MB, incorporated in liposomes, in ovarian-tumor-bearing embryos [74]. Fluorescence measurements revealed that topical application of liposomal MB resulted in higher fluorescence intensities in tumors compared with MB administered in solution [74]. Furthermore, cryosections of tumor samples were analyzed by fluorescence microscopy that demonstrated that MB-liposomes penetrated deeper into the tumoral tissues than free MB. For directly measuring drug distribution in CAM tissues after IV injection, the technique described by Lange et al. [19] has been

used in several papers evaluating DDS incorporating PS [20–23]. After IV administration of the PS, fluorescence microscopes are used to take pictures of the CAM vasculature at several timepoints after administration. From these fluorescent images, the extent of the fluorescence inside and outside blood vessels can be measured semiquantitatively. Then, the photographic contrast is calculated, and the extent to which the PS diffuses through CAM endothelial barrier is determined. Based on this technique, the PS pharmacokinetic profiles can be estimated. Avoiding extravasation of PS is a crucial issue for CNV-AMD treatment. Indeed, intravascular localization of PS is needed to decrease adverse effects on healthy neighboring tissues in the retina. The influence of the liposome composition on the circulation time of PS was highlighted with the CAM model. When THPC was administered as plain or PEGylated liposomes made of DPPC, THPC remained intravascular for at least 20 min postadministration [22]. On the contrary, higher extravasation of BPD-MA was observed during the same period, when loaded in liposomes made of dimyristoylphosphatidylcholine and egg yolk phosphatidylglycerol (Visudyne®) [19]. This observation suggests that DPPC liposomes, which were in a gel–solid state at body temperature, are more stable in the bloodstream than BPD-MA liposomes consisting of more fluid lipids. The differences in the intravascular residence time of THPP in solution or incorporated in PLGA NP were also revealed using this fluorescence technique [20]. After IV administration, NP remained intravascular for at least 25 min, whereas the nonencapsulated PS already leaked out of the blood vessels. Besides the physicochemical properties of the carrier, the nature of the PS itself influences the magnitude of PS extravasation from CAM blood vessels [21]. The encapsulation of TPP, TCPP, Ce6, and Pheo-a into PLA NP increased the residence time in blood vessels for all PS. Furthermore, the increase of the PS lipophilicity was shown to reduce the rate of extravasation. Measurements of CAM intravascular fluorescence allowed the estimation of pharmacokinetic profiles with several advantages over other methods, such as easy access to the CAM vasculature after opening the eggshell, the possibility of making measurements without tissue or blood sampling, and the pharmacokinetic profile calculation by imaging analyses rather than by chemical analyses. However, limitations of this approach include the difficulty to differentiate the fluorescence of the drug incorporated in the delivery system from the fluorescence of the released drug, and the impossibility to discriminate quantitatively between changes in PS concentration and fluorescence properties occurring during blood circulation. Thus, the pharmacokinetic profiles determined with the CAM model by fluorescence measurements are relative. 3.4.3. Biosensors Sampling of blood directly from chick embryos is restricted by the total volume of the blood. Consequently, the use of noninvasive methods for the analysis of drug concentration can be advantageous in this model. Biosensors are used to determine the concentration of substances by converting a biological response into an electrical signal [83]. The use of biosensors for acetaminophen [12,84] and glucose [85] has been evaluated with

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chick embryos. Acetaminophen sensors were placed on the top of the CAM of embryos at EDD 7 and allowed to incorporate into the CAM tissue for 1 week [12]. After IV administration, levels of acetaminophen in blood were determined with the biosensor, and blood samples were analyzed by HPLC in parallel. The results demonstrated that the current produced by the sensor reflected the change in blood levels of acetaminophen. Since the sensitivity of the biosensor is related to its contact with blood circulation, a device was added to induce local neovascularization around the biosensor location [84]. A fibrin matrix, containing modified chicken fibroblasts able to transfect the VEGF gene was formed on the surface of acetaminophen sensors [84]. This modification resulted in a massive increase in neovascularization, dramatically enhancing the sensor's function in vivo. Similar effects were observed with glucose sensors [85]. Although the chick embryos are not often used to perform pharmacokinetic and biodistribution studies, drug metabolism in various avian species and drug clearance in birds have been shown to be similar to that of mammals [86]. Indeed, birds can accomplish biotransformation reactions by functionalization and conjugation. Nevertheless, care should be taken to extrapolate from avian to mammalian models, because differences between the immune systems might change the fate of DDS. For instance, colloidal carriers IVadministered to mammals, such as liposomes, microparticles, and NP, are usually taken up by organs rich in the

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mononuclear phagocytic system, such as spleen and liver. The biodistribution of colloidal carriers in the chick embryo is still uncharacterized. 4. Advantages and limitations of chick embryos for evaluating drug delivery systems The interest in using chick embryos for the early evaluation of DDS has been outlined in this overview. However, the chick embryo is still a model and presents some advantages and limitations that are summarized in Table 4. The use of the chick embryo, as a whole living organism, overcomes some limitations encountered when working with simple in vitro systems frequently used in the pharmaceutical field. Although, in vitro drug release in simulated biological media and in vitro cellular models generate valuable data for characterizing DDS properties, these systems do not offer the full complexity of a living organism. Different administration routes used in mammals and humans can be used with the chick embryos, whereas the administration route parameter cannot be evaluated with in vitro models. Comparison of the genomes of chickens, mice and humans allows to get useful insights in the potential similarities and differences between these organisms [87,88]. Comparative genomics revealed that the chick genome is three times smaller than the one of both human and mouse, but contains approximately

Table 4 Advantages and limitations of chick embryos for the evaluation of drug delivery systems (DDS) Advantages

Limitations

General –Cheap and quick. –Complete in vivo environment. –Legal aspects. –Rapid screening of formulations. –Assessment of toxicity, biocompatibility and drug activity.

General –Lack of data enabling extrapolation to mammalian models. –Differences in drug metabolism and immune system with mammals.

Methodology –Transparent membrane. –Easy observation of DDS effects.

Methodology –Difficult choice of protocols and embryo age to perform experimentation. –Stimulation of angiogenesis by the carrier properties and by eggshell contamination. –Need of small amounts of DDS. –Short term evaluations (around 2 weeks from day 4-5 when CAM is formed until hatching). –Different administration routes (topical, IV, IP, etc). –DDS evaluation can be time-consuming (e.g. image analyses). –Continuous visualization of the DDS during the time of the experiment (topical –Limited amounts of tissue and blood. application). –Several samples can be tested on the same CAM. –Oral route cannot be tested. –Possibility of xenografting (e.g. tumors or other tissues). –Large pool of available techniques for embryo culture and response evaluation. –Rapid detection of possible side effects (mortality, embryotoxicity, and membrane irritation). –No need for surgical DDS implantation. –Easy tissue sampling compared with mammalian models. –No need of animal research facilities. Biology of chick embryo and CAM Biology of chick embryo and CAM –Well characterized biology and physiology of chick embryos. –Young chick embryos are not fully immunocompetent. –Presence of many mechanisms relevant for physiological and pathological –Rapid changes of CAM during embryonic development. angiogenesis and tumor growth. –Spontaneous vessel growth. –Different location of blood vessels within the CAM depending on the EDD and the vessel type. –CAM tissue response similar to mammalian response. –Selective transport of macromolecules through the CAM microvascular endothelium varies during development.

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the same number of genes [89–91]. Although about 60% of chicken genes have a single human orthologue, chicken and human orthologous genes exhibit lower sequence conservation (75.3%) than rodent and human orthologous genes (∼88%). Chicken genes involved in the basic structure and functions of the cells showed more sequence similarity with human genes than did those implicated in reproduction, immune response and adaptation to the environment [90]. For example, chickens use an avian specific family of keratins to produce scales, claws and feathers, whereas hair formation in mammals involves a distinct keratin family. Likewise, chickens are missing the genes involved in the production of milk proteins and tooth enamel [90]. Interestingly, interleukin-26 genes are present in both chickens and humans, whereas in mice and rats only a pseudogene is present [90]. Thus, chicken is the only known model organism to investigate the function of interleukin-26. Even if the genetic information is valuable, it is still difficult to establish to which extent the complex physiology of mammals and chick embryos are similar, and each physiological aspect must be compared case by case. For instance, some reviews compared the renal physiology [92], the biology of the pulmonary system [93], and the mechanism of body fluid regulation [94] of both birds and mammals. The production and function of red blood cells in vertebrate embryos have been reviewed as well [95]. In conclusion, assays with the chick embryo are economic and easy to perform when compared with mammalian models, but it is still difficult to translate the data to mammalian models and even to humans. 5. Conclusions and perspectives Although the chick embryo and its chorioallantoic membrane have been extensively used to study both angiogenesis and antiangiogenesis processes, its potential use has not been fully exploited in pharmaceutics and biopharmaceutics. Most of the DDS evaluated with the CAM model incorporated drugs modifying the vasculature, such as pro-, antiangiogenic molecules and PS. The strategy frequently used is to optimize the formulations in terms of excipients, drug loading, and other properties by in vitro testing. Then, chick embryos are used at the last step of the experimentation as a proof of concept, but in many studies, incomplete experimentation is done (lack of controls, few replicates, only one type of formulation is evaluated, subjective evaluation, etc). Other studies, particularly with antiangiogenic drugs, went further and used chick embryos as an intermediate step between in vitro tests and in vivo mammalian models. Qualitatively, some similarities were observed between CAM and mammalian models, but quantitative data showing the correlation between both models are still missing. Consequently, nowadays, it is difficult to extrapolate data derived form the CAM to other models. There are a large number of protocols for culturing embryos, administering formulations, and assessing the physiological or tissular responses. The choice of the protocol is directed by the DDS to be evaluated. One could question if the harmonization of these protocols would allow the validation of the assays performed with chick embryos, thus increasing their use in the pharmaceutical field. For instance, efforts have been made to

Fig. 5. Overview of available techniques for the evaluation of the chorioallantoic membrane. The effects of formulations can be assessed either by microscopy, or by molecular and biochemical analysis of the membrane. Microscopic observations of CAM can be done from the top of the membrane (en-face observation) or after cross-sectioning. These techniques have been already reviewed [6].

correlate the HET-CAM assay, a variation of the CAM assay for the evaluation of ocular irritation, with the Draize eye irritation test, performed in rabbits [96]. To improve DDS screening with the chick embryos, the pharmaceutical community can take advantage of the multiple methods developed for studying the body of chick embryos and the CAM. Available techniques for CAM evaluation have been reviewed [6], and are depicted in Fig. 5. Because of the low cost and simplicity of the assays with the chick embryo, this model offers the possibility of performing a high-throughput screening of DDS before using mammalian models, which are more expensive and need the approval of authorities. There is a great opportunity for using chick embryos in areas of growing interest in pharmaceutical research, such as antiangiogenic and photodynamic therapies for the treatment of cancer and diseases related to abnormal angiogenesis, wound healing therapies, tissue engineering, and new excipients research. References [1] C.R. Gardner, O. Almarsson, H. Chen, S. Morissette, M. Peterson, Z. Zhang, S. Wang, A. Lemmo, J. Gonzalez-Zugasti, J. Monagle, J. Marchionna, S. Ellis, C. McNulty, A. Johnson, D. Levinson, M. Cima, Application of high throughput technologies to drug substance and drug product development, Comput. Chem. Eng. 28 (2004) 943–953. [2] Nuffield Council on Bioethics, The context of animal research: past and present, The Ethics of Research Involving Animals, Nuffield Council on Bioethics, London, 2005, pp. 13–29. [3] Aristotle, Historia Animalium, vol. 2, Harvard University Press, Cambridge, 1970. [4] D. Ribatti, A. Vacca, L. Roncali, F. Dammacco, The chick embryo chorioallantoic membrane as a model for in vivo research on antiangiogenesis, Curr. Pharm. Biotechnol. 1 (2000) 73–82. [5] D. Ribatti, B. Nico, A. Vacca, L. Roncali, P.H. Burri, V. Djonov, Chorioallantoic membrane capillary bed: a useful target for studying angiogenesis and anti-angiogenesis in vivo, Anat. Rec. 264 (2001) 317–324. [6] M. Richardson, G. Singh, Observations on the use of the avian chorioallantoic membrane (CAM) model in investigations into angiogenesis, Curr. Drug Targets Cardiovasc. Haematol. Disord. 3 (2003) 155–185. [7] D. Ribatti, A. Vacca, G. Ranieri, S. Sorino, L. Roncali, The chick embryo chorioallantoic membrane as an in vivo wound healing model, Pathol. Res. Pract. 192 (1996) 1068–1076. [8] J. Borges, F.T. Tegtmeier, N.T. Padron, M.C. Mueller, E.M. Lang, G.B. Stark, Chorioallantoic membrane angiogenesis model for tissue engineering: a new twist on a classic model, Tissue Eng. 9 (2003) 441–450.

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