Drug Delivery Systems

1. Drug Delivery Systems

2. 基金委-董建华处长 高分子研究进展与国家自然科学基金近况 12月25日下午2:30，地点: 18层大楼1楼 院士系列学术报告: 颜德岳；江明；曹镛 高分子研究进展与国家自然科学基金近况 12月27日上午8:30，地点: 18层大楼1楼

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

4. Drug Delivery Systems (药物控制释放体系) • 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。

5. 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

7. Mechanisms of Polymer-based Drug Delivery Systems

8. I. Diffusion Mechanism • 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.

9. 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.

10. Examples • 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.

11. II. Solvent-Activated Mechanism • 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. Fig. c: osmotic system.

12. 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.

13. III. Chemical Reaction Mechanism • 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). Fig. d: polymeric drug conjugates.

14. Examples • 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).

15. Progress in Drug Delivery Systems

16. 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。

17. (A). Block-copolymer Nanospheres Scheme 1. Architecture of block-copolymer nanospheres which spontaneously form by self-assembly in water.

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

19. (B) Fig. Magentically controlled system.

20. 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。

21. (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。

22. Fig. pH or temperature controlled system.

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

24. 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

25. Gene Delivery/Therapy • Introduction • Progress in non-viral gene delivery • Prospects in gene delivery

26. I. Introduction • (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。

27. 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。

28. (B) Classification and Characteristics • 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.

29. Non-viral Vectors • 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.

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

31. (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).

32. 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.

33. Progress in non-viral gene delivery

34. 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.

35. I. Naked DNA delivery by physical method: to overcome safety issue and to realize efficient gene expression in vivo Figure 1 Overview of nonviral gene delivery technologies.

36. Table 2. Methods of non-viral gene transfer

37. 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.

38. 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.

39. II. Gene delivery using a chemical carrier: to establish functional gene delivery in vivo Novel carriers to achieve high-level gene expression and functional delivery have been designed. Gene carriers can be categorized into several groups: • 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.

40. (A) Lipid-mediated gene delivery • 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.

41. 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.

42. 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.

43. (B) Polymer-mediated gene delivery 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.

44. 1. Gene delivery process • 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.

45. 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.

46. 2. Other Polymer-based systems • 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.

47. 3. Control over DNA delivery • 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.

48. 4. Targeting of gene transfer • 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.

49. 5. Drawbacks and Modification • 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.

50. 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.