药物控制释放体系.ppt

　　药物控制释放体系 Drug Delivery Systems

　　Content

　　Introduction to Drug Delivery Systems (DDSs) Mechanisms of Polymer-based DDSs Progress in DDSs Gene Delivery and gene therapy

　　定义：药物控制释放是指在较长的时间内(至少12h)，按照预定速度向全身或某一特定器官连续释放一种或多种药物，并且在一段固定时间内，使药物在血浆和组织中的浓度能稳定某一适当水平(该浓度是使治疗作用尽可能大而副作用尽可能小的最佳水平)。而传统的给药方式(口服或注射)往往使血液中药物大幅度波动，即有时超过有效治疗指数而带来副作用，有时未达到有效治疗范围而失去疗效。 与传统的给药方式相比，药物控制释放具有以下潜在的优点： (1)可连续保持药物浓度在一个理想的疗效范围; (2)由于可靶向释放药物到某一特定细胞或组织而减少毒副作用; (3)可能减少所需药物剂量; (4)减少给药频率; (5)对于蛋白质和多肽药物，其体内半衰期短，可方便地进行药物释放而不至于失去药物活性。

　　Drug Delivery Systems (药物控制释放体系)

　　药物控释的途径： 经口(ingestion): 口服经胃肠道、消化道等;体腔内粘膜给药(眼内、口腔、舌下、鼻腔、直肠等) 注射(injection):动脉注射及静脉点滴给药;皮下及肌肉注射 透皮(transdermal) 发展阶段： 1950’s，传统型药物制剂 1950-70’s，缓释型药物制剂 1970’s，控释型药物制剂 1980’s，靶向型、智能型药物制剂

　　Mechanisms of Polymer-based Drug Delivery Systems

　　In some cases (a, reservoir systems), the drug is surrounded by a polymer membrane, such as a capsule or microcapsule. Fig. a: reservoir systems; b, matrix systems. In other cases (b, matrix systems), the drug is uniformly distributed through the system. In both cases, diffusion of the drug through the polymer backbone or pores in the polymer membrane is the rate-limiting mechanism.

　　I. Diffusion Mechanism

　　Release rates from membranes are determined by the steady-state Fick’s Law diffusion equation:

　　Here D is the concentration-independent drug diffusion coefficient in the membrane: 扩散系数(m2/s) J* is the drug molar flux：J为扩散通量 atoms/(m2·s)或kg/(m2·s) dc/dx: is the drug concentration gradient within the membrane.

　　Non-biodegradable polymer “Norplant” commodity: the silicone capsule containing contraceptives that are released by diffusion through polymer for 5 years. (Mr <400)---reservoir system (membrane-controlled diffusion); Ethylene-vinyl acetate (EVA), PSt, Ethyl-cellulose, Hydrogels (PVA)----matrix system (interconnecting pores); Biodegradable polymer PLGA system: combination of diffusion and polymer matrix degradation.

　　Examples

　　The osmotically controlled release system involves a tablet containing an osmotic agent surrounded by a semipermeable membrane (permeable to water but impermeable to salt or drug). The membrane contains a single laser-drilled hole. The external solvent, water, enters the tablet through the membrane at a constant rate and drives the drug out through the laser-drilled hole at a constant rate.

　　II. Solvent-Activated Mechanism

　　Fig. c: osmotic system.

　　An equation that describes release rates from these systems:

　　where K is a constant equal to the product of the membrane’s hydraulic permeability and its reflection coefficient, II is the osmotic pressure of the osmotic agent of the core formulation, C is the drug concentration inside the osmotic tablet core, and l is the membrane thickness. Examples: EVA, PMMA, PAA, Cellulose derivatives membranes.

　　In this case, water or enzymes cause degradation of a polymer which is used to encapsulate a drug (erodible or degradable system) or cleaves a bond between the drug and polymer, releasing the drug (pendant chain system).

　　III. Chemical Reaction Mechanism

　　Fig. d: polymeric drug conjugates.

　　From a chemical standpoint, bioerodible systems can be distinguished by three dissolution mechanisms: (1) water-soluble polymers insolubilized by degradable cross-links; (2) water-insoluble polymers solubilized by hydrolysis, ionization, or protonation of pendant side groups; and (3) water-insoluble polymers solubilized by backbone-chain cleavage to small water-soluble molecules. The most commonly used biodegradable polymer is poly(lactic acid) or lactic/glycolic copolymers (type 3). Others include poly(vinylpyrrolidine) (type l), copolymers of methyl vinyl ether (n-butyl half-ester) and maleic anhydride (type 2), poly(anhydrides) (type 3), poly(ortho esters) (type 3), poly(ecaprolactone) (type3), and poly(amino acids) (type 3).

　　Examples

　　Progress in Drug Delivery Systems

　　I. 靶向药物释放(Targeted Drug Delivery)或称部位导向释放(Site-specific Drug Delivery) 主动靶向：利用对药物制剂表面修饰的生物识别分子，如细胞-表面特异糖类、糖肽、糖酯、抗体(抗原)、酶等; 被动靶向：利用体系本身差异，如粒子大小、表面性质等影响其在体内的运行途径; 磁性导向：利用药物制剂具有的顺磁性，在服药后通过强磁场控制制剂的行径。

　　Scheme 1. Architecture of block-copolymer nanospheres which spontaneously form by self-assembly in water.

　　(A). Block-copolymer Nanospheres

　　Scheme 2. Schematic representation of the enhanced permeation retention model, which explains the selective accumulation of nanocarriers in the porous tumor tissue.

　　Fig. Magentically controlled system.

　　(B)

　　II. 自调节的药物释放(Self-Regulated Drug Delivery)

　　(A) 反馈控制药物释放(Feedback-Controlled Drug Delivery) 反馈控制药物释放体系是指对特定刺激物的浓度产生响应而释放药物的体内植入装置。 目前，最广泛研究的调节装置是葡萄糖响应胰岛素释放体系。 Kim和其合作者利用刀豆球蛋白A(ConA)与葡萄糖和糖基化胰岛素的竞争性和互补结合行为，系统研究了这一体系。其设想是将生物调谐与控制释放相结合，ConA为一外源性凝集素，对特异糖类的结合亲和性甚高。因此，可利用对硝基苯基糖衍生物使胰岛素糖基化，以提高ConA与胰岛素的结合性，这样可以防止低血糖条件下胰岛素的释放。

　　(B) 刺激敏感的药物释放(Stimuli-Sensitive Drug Delivery) 刺激敏感的药物释放是指能感知环境的变化并产生响应的药物释放。这些刺激主要是物理或化学信号。化学信号包括pH、代谢物及离子因素，它们将会改变体系中高分子链之间或高分子链与溶质之间的作用力。物理刺激包括温度或电势，它们将为分子运动提供能量并且改变分子间相互作用。 近来，人们发现含弱酸/碱基团的聚合物水凝胶，其溶胀体积随溶液 pH、离子强度而变化，从而影响介质对其扩散、渗透的能力。这种凝胶作为药物载体，可组成pH响应性药物释放体系。例如聚(甲基丙烯酸-2-羟基乙酯-共-甲基丙烯酸-2-二乙基氨基乙酯)共聚物。根据pH值变化，该体系能产生膨账或收缩而导致开-关机理来控制药物释放速度。 pH-敏感的高分子能用在靶向癌药物释放体系中，因为据报道癌细胞周围的pH低于正常细胞周围的 pH。这种pH 值的差异来自于癌细胞活跃的代谢功能或癌细胞表面存在的大量神经酸衍生物。

　　Fig. pH or temperature controlled system.

　　pH-sensitive Hydrogels

　　Fig. 4. pH-dependent ionization of polyelectrolytes. Poly(acrylicacid) and poly(N ,N-diethylaminoethyl methacrylate).

　　Fig. 5. Schematic illustration of oral colon-specific drug delivery using biodegradable and pH-sensitive hydrogels. 口服结肠定位给药系统

　　Introduction Progress in non-viral gene delivery Prospects in gene delivery

　　Gene Delivery/Therapy

　　(A) The basic concept of gene therapy is disarmingly simple — introduce the gene, and its product should cure or slow down the progression of a disease. 基因治疗可以定义为“把基因作为药物来治疗疾病”或“为达到治疗的目的,通过载体把核酸传送到病体”. 如果一位病人由于缺少某种已知基因而患病,那么把缺少基因通过一种特定的载体输送到病变细胞或组织内,使之表达,有可能会直接纠正基因缺乏,从而达到治愈疾病的目的;如果无法从基因的角度确定病人的病因,但其病理研究已十分清楚,那么可以利用载体把适当的基因或某些核酸类药物(如antisense oligonucleotides 或mRNA) 输送到病变细胞, 通过其他途径破坏该病的机制。

　　I. Introduction

　　自从1980年出现第一个关于哺乳动物基因转移的报告后，到1994年底，已有300多人参加了基因治疗的临床实验。体内的基因治疗对于一些人类疾病有着潜在的能力，如遗传性单一基因紊乱、复合性基因紊乱。

　　The vectors available now: the non-viral and viral vectors. These techniques are categorized into two general groups: naked DNA delivery by a physical method, such as electroporation and gene gun and delivery mediated by a chemical carrier such as cationic polymer and lipid. Viral vectors suffer from several drawbacks: a need for packaging cell lines (细胞系), problems with safety, toxicity, the elicitation of an immune response, the lack of cell-specific targeting, viral vector systems are rapidly cleared from the circulation, limiting transfection to ‘first-pass’ organs, such as the lungs, liver and spleen. Viral vectors have been implicated in the death of at least one patient, leading the suspension of clinical trials.

　　(B) Classification and Characteristics

　　Advantages of non-viral vectors they are easy to prepare and to scale-up, they are more flexible with regard to the size of the DNA being transferred, they are generally safer in vivo, they do not elicit a specific immune response and can therefore be administered repeatedly, they are better for delivering cytokine genes because they are less immunogenic than viral vectors. Disadvantages and Current Status less efficient in delivering DNA and in initiating gene expression, particularly when used in vivo. for this reason, few nonviral vectors have reached clinical trials, including naked DNA, DNA–cationic-LIPOSOME complexes (lipoplexes),DNA–polymer complexes and combinations of these.

　　Non-viral Vectors

　　Table 1. Non-viral vectors in gene therapy clinical trials

　　(C) Properties of the ideal gene therapy vector

　　Goals: the ideal gene delivery system should be specifically targeting, biodegradable, non-toxic, non-inflammatory, non-immunogenic and stable for storage. It should also have a large capacity for genetic material, efficient transfection and the capacity to be produced in high concentrations at low cost. Easy production The vector should be easy to produce at high titre on a commercial scale. (such as concentration technology for delivery in small volumes), and should have a reasonable shelf-life for transport and distribution. Sustained Expression The vector, once delivered, should be able to express its genetic cargo over a sustained period or expression should be regulable in a precise way. Different disease states have different requirements (for example, regulated expression in diabetes and lifetime expression in haemophilia，血友病).

　　Immunologically inert The vector components should not elicit an immune response after delivery. A humoral (体液) antibody response will make a second injection of the vector ineffective, whereas a cellular response will eliminate the transduced cells. Tissue targeting Delivery to only certain cell types is highly desirable, especially where the target cells are dispersed throughout the body, or if the cells are part of a heterogeneous population (such as in the brain). Size capacity The vector should have no size limit to the genetic material it can deliver. The coding sequence of a therapeutic gene varies from 350 base pairs for insulin, to over 12,000 base pairs for dystrophin(营养不良). Replication, segregation or integration The vector should allow for site-specific integration of the gene into the chromosome of the target cell, or should reside in the nucleus as an episome (附加体, 游离体, 游离基因); that will faithfully divide and segregate on cell division.

　　Progress in non-viral gene delivery

　　Naked DNA delivery by physical method: to overcome safety issue and to realize efficient gene expression in vivo; Gene delivery using a chemical carrier: to establish functional gene delivery in vivo; Nonviral vector modifications with peptides to increase intracellular gene delivery; Reduction of immune responses by modifying the administration protocol or the composition of the DNA; Design of tissue-specific, self-replicating and integrating plasmid expression systems to facilitate long-lasting gene expression.

　　Progress in non-viral gene delivery

　　Figure 1 Overview of nonviral gene delivery technologies.

　　I. Naked DNA delivery by physical method: to overcome safety issue and to realize efficient gene expression in vivo

　　Table 2. Methods of non-viral gene transfer

　　Electroporation (电穿孔) The application of controlled electric fields to facilitate cell permeabilization, is used for enhancement of gene uptake into cells after injection of naked DNA. In addition, electroporation can achieve long-lasting expression and can be used in various tissues. Skin is one of the ideal targets because of the ease of administration. Gene gun Gene gun can achieve direct gene delivery into tissues or cells. Shooting gold particles coated with DNA allows direct penetration through the cell membrane into the cytoplasm and even the nucleus, bypassing the endosomal compartment. Ultrasound Ultrasound can increase the permeability of cell membrane to macromolecules such as plasmid DNA. Indeed, enhancement of gene expression was observed by irradiating ultrasonic wave to the tissue after injection of DNA. Since ultrasound application is flexible and safe, its use in gene delivery has a great advantage in clinical use.

　　Hydrodynamic injection Hydrodynamic injection, a rapid injection of a large volume of naked DNA solution (eg 5 mg plasmid DNA injected in 5–8 s in 1.6 ml saline solution for a 20 g mouse) via the tail vein, can induce potent gene transfer in internal organs, especially the liver. Blood Occlusion Significant gene expression can be achieved in the liver by transiently restricting blood flow through the liver immediately following peripheral intravenous injection of naked DNA. Occlusion of blood flow either at vena cava or at hepatic artery and portal vein increased the expression level in the liver. Presumably, the injected DNA is internalized into the hepatic cells by receptor-mediated mechanism as proposed by Budker et al or via a nonreceptor-mediated pathway.

　　II. Gene delivery using a chemical carrier: to establish functional gene delivery in vivo

　　those forming condensed complexes with the DNA to protect the DNA from nucleases and other blood components; those designed to target delivery to specific cell types; those designed to increase delivery of DNA to the cytosol or nucleus; those designed to dissociate from DNA in the cytosol; those designed to release DNA in the tissue to achieve a continuous or controlled expression. Lipids and polymers are mainly used for gene delivery.

　　Novel carriers to achieve high-level gene expression and functional delivery have been designed. Gene carriers can be categorized into several groups:

　　Liposome-based gene delivery, first reported by Felgner in 1987, is still one of the major techniques for gene delivery into cells. In 1990s, a large number of cationic lipids, such as quaternary ammonium detergents, cationic derivatives of cholesterol and diacylglycerol, and lipid derivatives of polyamines, were reported. However, the development of novel types of lipid molecules appears to be saturated, and most of the efforts have shifted to improving efficacy by the modification listed above, as well as to specific in vivo applications.

　　(A) Lipid-mediated gene delivery

　　Fig. 1. Cationic-lipid–DNA complexes. Cationic lipids and DNA are mixed to form complexes that can enter cells by endocytosis. Once inside the cell, the DNA is released and transported to the nucleus.

　　Cationic-liposome-mediated gene transfer has, however, been successfully used in vitro and in vivo in gene therapy experimental models, and has also been evaluated in several clinical protocols. In several phase-I human trials, direct in vivo injection of a pDNA–lipid complex expressing the major-histocompatibility (组织相容性)-complex-class-I gene, HLA-B7, produced a clinical response in HLA-B7-negative melanoma patients. An interleukin [白(细胞)介素,白细胞间素]-2-expressing pDNA–lipid complex was evaluated in a phase-I and -II trial of patients with melanoma (黑素瘤), sarcoma (肉瘤) or renal cell carcinoma.

　　Fig. 2. Structure of a cationic polymer. Poly-L-lysine (PLL) is shown as a representative example. PLL is a linear, biodegradable molecule that can be modified easily.

　　(B) Polymer-mediated gene delivery

　　Like cationic lipids, cationic polymers such as poly-L-lysine (PLL) derivatives,and polyethyleneimine, polyamidoamine and polymethacrylate dendrimers, form electrostatic complexes with the negatively charged DNA. These complexes can be taken up by cells. For successful transfection, a plasmid must be delivered to the nucleus, a process that requires cellular uptake of polymer–DNA (polyplexes) or lipid–DNA complexes. This is most likely to occur via endocytosis, followed by endosomal escape and transport to the nucleus. A DNA–cationic-carrier complex requires endosomal and/or lysosomal release because it is entrapped in these organelles after its cellular uptake. The polyplex or lipoplex must dissociate, either in the cytosol or in the nucleus, and this might be a crucial step in the transfection process.

　　1. Gene delivery process

　　Fig. 2. Current systems are invariably taken up into endosomes where they would eventually be degraded. After escaping into the cytoplasm (胞质) the nucleic acid (plasmid DNA) needs to gain entry into the nucleus to be able to utilise the nuclear transcription machinery and initiate gene expression. Access to the nuclear machinery can in principle occur during cell division when the nuclear envelope disappears through the nuclear pores which allow shuffling of suitable molecules between nucleus and cytoplasm.

　　Polymer-based systems (e.g. using collagen, lactic or glycolic acid, polyanhydride or polyethylene vinyl coacetate) provide several potential advantages for the therapeutic delivery of DNA (or of drugs). First, DNA encapsulation within the polymer can protect against degradation until release. Second, injection or implantation of the polymer into the body can be used to target a particular cell type or tissue. Third, drug release from the polymer and into the tissue can be designed to occur rapidly (a bolus delivery) or over an extended period of time; Thus, the delivery system can be tailored to a particular application. The choice of polymer and its physical form determine the time-scale of release.

　　2. Other Polymer-based systems

　　Control over DNA delivery can be achieved by the formation of both synthetic and natural polymers in a variety of geometries and configurations, such as reservoirs, matrices, and microspheres. Microspheres (or pellets) can be delivered in a minimally invasive manner (e.g. by direct injection or by oral delivery), Matrices can be implanted at the appropriate site, for example, for applications in tissue repair and wound healing.

　　3. Control over DNA delivery

　　Targeting of gene transfer has also been achieved by modification of gene carriers using cell targeting ligands, such as asialoglycoproteins for hepatocytes (肝细胞), anti-CD3 and anti-CD5 antibodies for T cells, transferrin (转运蛋白) for some cancer cells, insulin, or galactose. In addition, a targeted folate-expressing, cationic-liposome-based transfection complex has been shown to specifically transfect folate-receptor-expressing cells and tumours, suggesting that this is a potential therapy for intraperitoneal (腹膜内的) cancers.

　　4. Targeting of gene transfer

　　However, intrinsic drawbacks with cationic carriers, such as solubility, cytotoxicity and low transfection efficiency, have limited their use in vivo. These vectors sometimes attract serum proteins and blood cells when entering the circulation, resulting in dynamic changes in their physicochemical properties. Polyamidoamine and polyethyleneimine dendrimers have a high transfection efficiency in vitro and in vivo, but are cytotoxic and have low solubility when complexed with DNA. The attachment of polyethylene glycol to PLL provides a biocompatible protective coating for the DNA complex.

　　5. Drawbacks and Modification

　　More complex gene transfer systems use cationic amphiphiles, such as polymeric polyethylimines, polyamidoamine ‘starburst’ dendrimers, polylysine conjugates and cationic liposomes, which can be combined with naked DNA, mRNA or larger DNA fragments to produce complex particles. Polycation addition leads to electrostatic neutralization of anionic charges, and condenses the polynucleotide structure thereby protecting it against nuclease digestion. pDNA and cationic amphiphiles can be formulated in different ratios to produce complexes of diverse size and surface-charge properties. Additionally, complexes bearing a net positive charge display enhanced binding to negatively charged cell membranes, leading to increased cellular uptake.

　　Fig. 3. Viral–non-viral hybrid vectors. DNA is bound to a poly-L-lysine (PLL)–transferrin conjugate (a) to form a PLL–transferrin–DNA complex (b). Transferrin (red) binds to specific receptors on the surface of some cancer cells, thereby targeting gene delivery to these cells (c). Inactivated adenovirus (腺病毒)particles (blue) are added to this complex, possibly aiding entry into the cell and protecting the DNA against endosomal degradation.

　　(C) Viral–non-viral hybrid vectors

　　Hybrid vectors incorporate viruses or viral peptides into traditional cationic-amphiphile-based vector systems. The efficiency of synthetic vectors can be improved using artificial nucleic-acid carriers incorporating functional elements that mimic viruses. For example, the adenovirus hexon protein enhances the nuclear delivery and increases the transgene expression of polyethyleneimine–pDNA vectors. Non-viral vectors have also been designed to mimic the receptor mediated cell entry of adenoviruses; Formulations that combine the merits of both viral and non-viral systems, such as a virus–cationic-liposome–DNA complex, ‘haemagglutinating virus of Japan’ liposomes, and cationic-lipid–DNA mixed with the G glycoprotein from the ‘vesicular stomatitis virus’ envelope, have been developed.

　　Progress in hybrid vector

　　(D) Peptide-based gene delivery system

　　Amphiphilic a-helical peptides, containing cationic amino-acids, can be used as gene carriers into cells. These peptides are readily available, owing to recent developments in production methods, allowing the design and synthesis of functional gene carrier molecules, such as carbohydrate-modified peptides, for targeted gene delivery. Furthermore, the use of peptide-based gene carriers enables the construction of well-defined molecules, which cannot be achieved using polymer-based carriers.

　　Novel polymeric delivery systems (e.g. nanospheres), which can be administered in novel ways (e.g. aerosols,气溶胶), are being developed. The smaller the size of the condensed DNA particles, the better the in vivo diffusion towards target cells and the trafficking within the cell. Individual plasmid molecule can be collapsed to a nanoparticle using designed detergents. For example, in mice, nanoparticle-based gene delivery, targeted to the neovasculature using an integrin-targeting ligand, resulted in tumour regression.

　　(E) Nanoparticle-based gene delivery system

　　The association of non-viral gene vectors with supramagnetic nanoparticles, targeted by application of a magnetic field, increased efficacy by up to several-hundred-fold. The high TRANSDUCTION efficiency observed in vitro was reproduced in vivo using magnetic-field-guided local transfection in the gastrointestinal (胃与肠的) tract and in blood vessels.

　　(F) A physical and chemical combination: magnetofection

　　Physical techniques for gene delivery into cells such as electroporation, with and without adjuvants (佐剂), will be significantly optimized; Knowledge of the interaction of naked DNA with serum components and cell surface receptors will continue to accumulate. Immune responses originating from CpG motifs and nonviral gene carriers will diminish; The structure of gene carriers will be further optimized and tailored for specific uses such as systemic administration, local injection or organ-specific delivery; Novel ligands for targeted delivery of DNA will be found; Translocation mechanisms for plasmid DNA within the cell will be identified – these may provide novel strategies for efficient delivery; More tissue-specific, site-specific integrating or self-replicating plasmid vectors are likely to appear.

　　Prospects in gene delivery

　　Introduction Polymer scaffolds for tissue engineering Progress in tissue engineering

　　Tissue Engineering

　　Fig. 1. UNOS organ transplant statistics for 1990 to 1999 [6] documenting the wait-listed patients (O) and transplants(●).

　　Introduction

　　1980s, R&D in tissue engineering and biomaterials took off. As part of this interest, several biomedical engineering departments were established at major universities around the world. 1987 年春美国自然科学基金工程理事会在研讨生物工程前景时, 确立了“组织工程”这一概念。1988 年在美国L ake Tahoe举行的专家小组会上首次确定了“组织工程”的定义, 从而明确了“组织工程”的研究范围和目标。同年美国国家科学基金会受理和资助了组织工程方面的研究项目。 1995～ 1999 年间组织工程方面的论文达32684 篇, 涉及人体的各种组织。国际刊物“组织工程”也于1995 年创刊。 据估计组织工程潜在市场大约是4000 亿美元。目前组织工程研究已涉及到的组织有肝、心脏、胰腺、神经、血管、角膜、皮肤、韧带、软骨和硬骨等。

　　组织工程：利用工程学和生命科学的基本原理, 开发能恢复、维持或改善受损组织或器官功能的生物代替物。 因此，组织工程综合了细胞生物学、工程学、材料学和临床医学领域, 用活细胞和细胞外基质或骨架构造一个新的功能化组织或器官。 研究内容：种子细胞、生物材料、构建组织和器官的方法与技术、以及 组织工程的临床应用研究。 基本方法：将体外培养的高浓度的正常组织细胞扩增后吸附于一种生物相容性良好并可被机体降解吸收的生物材料上,形成具有三维空间结构的复合体。然后将这种细胞- 生物材料复合体植入组织器官的病损部位, 种植的细胞在生物材料被机体逐渐降解吸收过程中继续生长繁殖,形成新的具有相应形态和功能的组织和器官,达到修复创伤和重建功能的目的。

　　Figure 1. Schematic illustration of typical tissue engineering approaches. Cells are obtained from a small biopsy from a patient, expanded in vitro, and transplanted into the patient either by injection using a needle or other minimally invasive delivery approach, or by implantation at the site following an incision (cut) by the surgeon to allow placement.

　　Typical tissue engineering approaches

　　Polymer scaffolds in tissue engineering

　　I. Polymer Scaffolds Tissues or organs can be potentially engineered with a number of different strategies, but a particularly appealing approach utilizes a combination of a patient’s own cells combined with polymer scaffolds. A variety of tissues are being engineered using this approach including fabricated artery, bladder, skin, cartilage, bone, ligament, and tendon. Several of these tissues are now at or near clinical uses. II. Polymeric Hydrogels for Scaffolds An exciting alternative approach to cell delivery for tissue engineering is the use of polymers (i.e., hydrogels) that can be injected into the body. This approach enables the clinician to transplant the cell and polymer combination in a minimally invasive manner.

　　在组织工程中骨架起中心作用, 它不仅为特定的细胞提供结构支撑作用, 而且还起到模板作用, 引导组织再生和控制组织结构。 骨架的具体作用如下: (1) 在植入时骨架可引导细胞到预定位置, 给工程化组织限定有限空间, 引导组织再生过程。虽然分离出的细胞可以直接注入体内, 但不能形成有效的新组织; (2) 大多数哺乳动物细胞是固着型细胞, 如不给它们提供附着基质就会死去。因此, 基质骨架致关重用; (3) 基质或骨架的形貌能引导再生组织的结构, 如尺寸和形貌等, 故间接影响再生组织的功能; (4) 理想的基质骨架能引导特殊的细胞功能, 引导和调节细胞间的相互作用; (5) 聚合物骨架提供机械支撑作用, 以抗击压力等外力, 在人体中维持组织形状和骨架完整性。 (6) 高分子骨架还可构成宿主免疫系统分子的物理障碍, 避免人体免疫反应;

　　A. 组织工程中骨架的重要作用

　　组织工程多孔支架需要满足以下要求: 良好的生物相容性,即无明显的细胞毒性、炎症反应和免疫排斥; 合适的可生物降解吸收性, 即与细胞、组织生长速率相适应的降解吸收速率; 合适的孔尺寸、高的孔隙率( > 90 %) 和相连的孔形态,以利于大量细胞的种植、细胞和组织的生长、细胞外基质的形成、氧气和营养的传输、代谢物的排泄以及血管和神经的内生长; 特定的三维外形以获得所需的组织或器官形状; 高的表面积和合适的表面理化性质以利于细胞粘附、增殖和分化,以及负载生长因子等生物信号分子; 与植入部位组织的力学性能相匹配的结构强度,以在体内生物力学微环境中保持结构稳定性和完整性, 并为植入细胞提高合适的微应力环境。

　　B. 组织工程多孔支架

　　组织工程多孔支架的孔形态主要有纤维(网)、多孔海绵或泡沫、相连管状结构等三种,相应地,其致孔方法和技术也各不相同。 纤维网：是由纤维构成的无纺布或者是由纤维编制成的孔径可变更的三维骨架。 这种骨架的优点是表面积大, 有利于细胞粘附和养分的扩散, 因此对细胞存活和生长有利; 缺点是骨架结构稳定性不好。 一种方法是纤维固定技术, 例如将PLA 溶液喷到PGA 网上, 溶剂挥发后PLA 镶嵌在网络上, 加热使PGA 熔化, PGA 纤维在搭接处被焊接在一起。冷却后PLA 被溶剂溶解掉。用此方法PGA 纤维不经任何化学和形状变化而被焊接在一起, 结构得到稳定。 另一种方法是将PLA 溶液以雾状喷在网的表面, 溶剂挥发后形成一涂层。这种复合结构综合了纤维的力学性能和PLA 的表面特性。

　　C. 组织工程多孔支架的孔形态

　　图2　PGA 网络镶嵌上PLA 的电镜照片

　　多孔泡沫或海绵支架的致孔方法主要有粒子致孔法、相分离法、气体发泡法和烧结微球法等。 粒子致孔法：将组织工程材料和致孔剂粒子制成均匀的混合物,然后利用二者不同的溶解性或挥发性,将致孔剂粒子除去,于是粒子所占有的空间变为孔隙。 致孔剂粒子可采用氯化钠、酒石酸钠和柠檬酸钠等水溶性无机盐或糖粒子,也可用石蜡粒子或冰粒子。 最常用的方法是,利用无机盐溶于水而不溶于有机溶剂、聚合物溶于有机溶剂而不溶于水的特性,用溶剂浇铸法将聚合物溶液/ 盐粒混合物浇铸成膜,然后浸出粒子得到多孔支架。该法通常称为溶剂浇铸/ 粒子浸出法 (solution casting/ particulate leaching)。 由Mikos 等作为纤维连结法的改进而提出,已成功地用于软骨细胞的培养和软骨组织的生成。粒子浸出法制得的多孔支架的孔隙率可达91～93 % ,孔隙率由粒子含量决定,与粒子尺寸基本无关;孔尺寸50～500μm ,由粒子尺寸决定,与粒子用量基本无关;孔的比表面积随粒子用量增大和粒径减小而增大,变化范围为0. 064～0. 119μm- 1 。

　　相分离法/ 冷冻干燥法：指将聚合物溶液、乳液或水凝胶在低温下冷冻,冷冻过程中发生相分离,形成富溶剂相和富聚合物相,然后经冷冻干燥除去溶剂而形成多孔结构的方法。因而,相分离法又往往称为冷冻干燥法,按体系形态的不同可简单地分为乳液冷冻干燥法、溶液冷冻干燥法和水凝胶冷冻干燥法。

　　图4　相分离法制备的PLA 电镜照片

　　Figure 1. SEM photomicrographs of cross sections of PLLA sponges prepared with the weight fractions of ice particulates of 70% (a), 80% (b). The pore shapes were almost the same as those of the ice particulates. The degree of interconnection within the sponges increased as the weight fraction of the ice particulates increased.

　　II. Polymeric hydrogels for Scaffolds

　　Hydrogels in tissue engineering must meet a number of design criteria to function appropriately and promote new tissue formation. These criteria include both classical physical parameters (e.g., degradation and mechanics) as well as biological performance parameters (e.g., cell adhesion). The mechanical properties of hydrogels are important design parameters in tissue engineering, as the gel must create and maintain a space for tissue development. The interactions of cells with hydrogels significantly affects their adhesion as well as migration and differentiation. An absolutely critical parameter is the biocompatibility of hydrogels.

　　A. Design Parameters for Hydrogels in Tissue Engineering

　　Alginate is a well-known biomaterial obtained from brown algae and is widely used for drug delivery and in tissue engineering due to its biocompatibility, low toxicity, relatively low cost, and simple gelation with divalent cations such as Ca2+, Mg2+, Ba2+, and Sr2+ (Figure 5a). Alginate has found uses to date as an injectable cell delivery vehicle as well as wound dressing, dental impression, and immobilization matrix. Alginate gel beads have also been prepared and used for transplantation of chondrocytes, hepatocytes, and islets of Langerhans to treat diabetes. Alginate itself may not be an ideal material because it degrades via a process involving loss of divalent ions into the surrounding medium, and subsequent dissolution. This process is generally uncontrollable and unpredictable. Therefore, covalent cross-linking with various types of molecules and different cross-linking densities has been attempted to precisely control the mechanical and/or swelling properties of alginate gels (Figure 5b-d).

　　B. Example: Alginate

　　Figure 5. Chemical structure of (a) sodium alginate and various cross-linking molecules used in covalent crosslinking reactions, including (b) adipic acid dihydrazide, (c) L-lysine, and (d) poly(ethylene glycol)-diamine. Alginate can be oxidized with sodium periodate under mild reaction conditions to infer main chain lability to hydrolysis as well (e).

　　Another potential limitation in using alginate gels in tissue engineering is the lack of cellular interaction. Alginate is known to discourage protein adsorption due to its hydrophilic character, and it is unable to specifically interact with mammalian cells. Therefore, alginate has been modified with lectin, a carbohydrate specific binding protein, to enhance ligand-specific binding properties. An RGD-containing cell adhesion ligand has also been covalently coupled to alginate gels to enhance cell adhesion. These modified alginate gels have been demonstrated to provide for the adhesion, proliferation, and expression of differentiated phenotype of skeletal muscle cells (Figure 6).

　　Figure 6. Myoblast (成肌细胞) adhesion onto (a) unmodified and (b) GRGDY-modified alginate hydrogels. Very few cells adhere to unmodified alginate gels, while cells readily adhere, spread, and function on the modified gels.

　　Progress in tissue engineering

　　I. Genetically Engineered Polypeptides Hydrogels

　　In brief, one may insert DNA templates of predetermined sequences into the genome of bacteria and produce polypeptides with predetermined structure and controlled properties. This method enables one to design and engineer various sequences of polypeptides with known functions, including elasticity, stiffness, degradation, and cellular interactions. Silk-like polypeptides have been prepared by this technique, and a Gly-Ala-rich sequence has been introduced into these artificial proteins to form reversible hydrogels in response to environmental changes of pH or temperature.

　　Elastin-mimetic polypeptides, comprised of a Gly-Val-Pro-Gly-any amino acid sequence, have also been studied and considered to have potential for artificial extracellular matrices in tissue engineering. This technique is not appropriate to economically produce biomaterials in large scale at the current time, and one is also unable to easily modify the polymer product as any change requires re-engineering of the entire system.

　　Another critical issue in the design of hydrogels for tissue engineering is that many tissues (e.g., bone, muscle, and blood vessels) exist in a mechanically dynamic environment. Many current hydrogels do not possess appropriate mechanical properties for these mechanically dynamic environments. It has been previously demonstrated that mechanical signals result in alterations of cellular structure, metabolism, and transcription and/or translation of various genes. The gels must appropriately convey the mechanical signals to these incorporated cells. It have recently reported that mechanical signals may be exploited to control growth factor release from hydrogels, and this could provide a novel approach to guide tissue formation in mechanically stressed environments.

　　II. Appropriate mechanical properties of hydrogels

　　Fig. 5. VEGF release from alginate scaffolds resulting in angiogenesis: (a) with mechanical stimulation of alginate gels and (b) without mechanical stimulation of alginate gels. Arrows indicate blood vessel formation in the muscle tissue surrounding the implanted gels.

　　Fig. A microfabricated bioreactor for perfusing 3D liver tissue engineered in vitro. (A) A cross section showing tissue aggregates growing attached to the inside walls of the narrow channels of the silicon-chip scaffold. (B) A bioreactor containing a 0.2-mm-thick silicon-chip scaffold etched with 0.3-mm-diameter channels. (C) Hepatocytes seeded onto the scaffold of the bioreactor attach to the walls of the channels and reorganize to form 3D structures that are reminiscent of liver. (D) Scanning electron micrograph showing vessel-like structures assembled from endothelial cells at the fluid-tissue interface in the bioreactor channels.

　　III. Tissue-Engineered Model Systems

　　Tissue engineering can be applied to the development of drugs to treat many diseases that could be prevented or even cured if such drugs were available today. The greatest impact of tissue engineering in the coming decade will be for designing in vitro physiological models to study disease pathogenesis and for developing molecular therapeutics. For example, the creation of tissues containing hierarchical cell-cell interactions under appropriate mechanical stresses (including perfusion shear as found in the microcirculation) will take in vitro systems even closer to living tissues.

　　人工皮肤是发展较快的一个领域。体外制造人工皮肤已不再是一个技术难题, 目前已有数种产品应用于临床治疗。二度以上的烧伤和慢性溃疡等, 应用组织工程皮肤技术有很好的疗效。 人工皮肤基本上可分为三个大的类型:表皮替代物、真皮替代物和全皮替代物。目前, 表皮替代物由生长在可降解基质或聚合物膜片上的表皮细胞组成。真皮替代物的基础是二倍体成纤维细胞的培养, 含有活细胞或不含细胞成分的基质结构, 用来诱导成纤维细胞的迁移、增殖和分泌细胞外基质。而全皮替代物包含以上两种成分, 既有表皮结构又有真皮结构。 人工复合皮肤替代物虽然包含了表皮与真皮两层结构, 但存在一个共同的问题: 即缺乏毛囊、汗腺和皮脂腺等皮肤附属器。因此,组织工程化皮肤缺少附属器是有待于解决的关键问题之一, 是今后皮肤组织工程研究的一个方向。

　　IV. 皮肤组织工程

　　In 1981, a skin equivalent consisting of a silicone cover over a sponge of porous collagen cross-linked with chondroitin was used successfully to treat severe burns. In this decade, several products reached the market. In 1996, Integra’s Artificial Skin was approved for as an in vivo, nonbiological tissue regeneration product. In 1997, Apligraf, produced by Organogenesis, is the first manufactured living human organ, specifically multilayered skin, to be recommended for approval by an advisory panel to the FDA. Apligraf was approved for the treatment of venous leg ulcers in Canada, and was launched there in August 1997 by NovartisPharmaceuticals Canada (Dorval, Canada). In 1998, the General and Plastic Surgery Devices Advisory Panel to the US Food and Drug Administration recommended unconditional approval of Apligraf (Graftskin) Human Skin Equivalent for the treatment of venous leg ulcers.

　　Figure. Regeneration of two-dimensional (skin) tissues using stem cells. Skin autografts are produced by culturing keratinocytes (角化细胞) under appropriate conditions not only to generate an epidermal sheet, but also to maintain the stem cell population. The epidermal sheet is then placed on top of a dermal substitute comprising devitalized dermis or bioengineered dermal substitutes seeded with dermal fibroblasts.

　　血管组织工程: 血管疾病是世界上发病率最高的疾病之一, 其主要的治疗手段是血管移植术。有资料表明, 每年仅美国的血管移植手术就超过140 万例。 目前血管组织工程的热点在于制备管径小于6 mm 的小血管。 以前小血管移植失败的原因, 在急性期主要为血栓形成; 在慢性期主要为平滑肌细胞向移植物管腔内增生以及吻合处形成血管翳导致管腔阻塞。 经过长期的探索, 发现血管内皮细胞是保持血管稳定性的天然调节物, 在血管损伤后内皮细胞单层具有抗血栓形成和抑制平滑肌细胞增生的作用, 并且在人和动物的血管移植模型中显示出提高血管通畅率的能力。

　　V. 血管组织工程

　　A critical future challenge facing this field is how polymers may be used to promote blood vessel network formation in the tissue. One important approach to actively modulate the vascularization process is the local delivery of either angiogenic factors or blood vessel forming cells to the engineered site using hydrogels. Examples: Various growth factors including vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), and bone morphogenetic protein (BMP) could be incorporated into hydrogels depending on the desired tissue type. Delivery of plasmid DNA containing genes encoding the angiogenic proteins may be another approach to enhance vascular network formation in engineered tissues. Co-transplantation of endothelial cells, which comprise blood vessels, along with the primary cell type of interest may allow one to rapidly form blood vessels in an engineered tissue.

　　Promoting the formation of blood vessel network

　　Fig. Schematic illustration of blood vessel formation promoted by including growth factors (a) or by seeding endothelial cells (d) into the polymer scaffold. Growth factors encourage existing blood vessels in the surrounding host tissue to grow into the scaffold (b), and the transplanted endothelial cells will form new blood vessels within the scaffold and grow outward toward the host tissue (e). Ultimately, new vessels combine with existing blood vessels to create functional blood vessels capable of blood flow.

　　骨缺损是临床常见的疾患, 骨移植已成为临床上仅次于输血的组织移植手术。组织工程学技术为骨缺损的修复提供了新的方法, 其研究方法主要分为两种: ①将载体材料与成骨因子在体外复合, 然后植入体内, 通过成骨因子的作用诱导种子细胞向成骨细胞方向分化, 进而形成新骨; ②将细胞在体外培养, 获得足够数量的成骨细胞, 并与载体材料在体外组装后植入骨缺损部位。 生物活性陶瓷是目前广泛应用的骨替代材料之一 。这些材料包括羟基磷灰石、双相羟基磷灰石、生物活性玻璃陶瓷等, 有良好的生物相容性, 耐磨, 弹性模量接近骨骼, 可制成多孔结构。通过加入金属纤维或与生物高分子材料复合物等增加韧性、强度的处理, 可具有常规陶瓷不可比拟的优点, 如强度高、韧性好、表面光洁等。 骨形成是一个十分复杂的过程, 在众多的影响因素中生长因子的作用十分重要。目前, 在已经确定对骨形成有明显作用的生长因子有骨形态发生蛋 白(BMP) 、胰岛素样生长因子( IGF) 、转化生长因子-β(TGF -β) 、碱性成纤维细胞生长因子(bFGF) 以及血小板衍生生长因子(PDGF) 等。这些生长因子在骨修复中起着促进细胞分裂、增殖、迁移和促进基因表达的作用。

　　VI. 骨组织工程

　　Figure. Regeneration of three-dimensional (bone) tissues using stem cells. Bone regeneration requires ex vivo expansion of marrow-derived skeletal stem cells and their attachment to three-dimensional scaffolds, such as particles of a hydroxyapatite/tricalcium phosphate ceramic. This composite can be transplanted into segmental defects and will subsequently regenerate an appropriate three-dimensional structure in vivo.

　　近年来, 国内外心肌组织工程研究取得了明显进展, 展现了良好的临床应用前景。目前主要采用两条途径: ①直接将细胞种植到心肌内; ②通过将细胞接种到可降解支架材料上, 在体外再造出心肌组织。 目前直接移植入心肌的细胞主要有三大类:同种异体细胞、转基因细胞和自体细胞。同种异体或转基因来源的细胞常用人胚胎干细胞、异基因心肌细胞以及转基因新生儿和胎儿心肌细胞等。 人源的胚胎干细胞在治疗疑难疾病上具有巨大的潜力, 除了可用于治疗心功能衰竭、帕金森病和阿尔茨默病之外, 还可作为发展基因治疗药物以及研究人早期胚胎形成的重要工具。 心肌组织工程是一个相对较新的领域, 目前关于体外构建具有三维结构的心肌组织的研究报道较少。

　　VII. 心肌组织工程