Cancer Therapy Vol 3, 131-138, 2005

Micro and nano drug delivery systems in cancer therapy

Review Article

Gorka Orive, Rosa Mar£a Hern΅ndez, Alicia R. Gasc½n, José Luis Pedraz*

Laboratory of Pharmacy and Pharmaceutical Technology, Faculty of Pharmacy, University of the Basque Country, Vitoria–Gasteiz, Spain

__________________________________________________________________________________

*Correspondence: Dr. José Luis Pedraz, Professor of the Laboratory of Pharmacy and Pharmaceutical Technology, Faculty of Pharmacy, University of the Basque Country, Vitoria -Gasteiz. Spain; Phone: +34 945-013091; Fax: +34 945-013040; E-mail: knppemuj@vc.ehu.es

Key words: Micro and nano drug delivery systems, Micro and nanotechnologies, cancer therapy, Macromolecular conjugation

Abbreviations: blood-brain barrier, (BBB); Food and Drug Administration, (FDA); leuteinizing-hormone-releasing hormone, (LHRH); permeability and retention effect, (EPR); polyethylene glycol, (PEG); poly(lactic-co-glycolic), (PLGA); recombinant human growth hormone, (rhGH); tissue inhibitor of metalloproteinase, (TIMP)

Received: 23 February 2005; Accepted: 1 March 2005; electronically published: March 2005

Summary

The delivery of any drug at the right time and in the target where it is needed and at the level that is required is essential to realize the full potential of therapeutic molecules. These requirements are already more important in the case of cancer chemotherapies due to their high toxicity which could lead to serious side effects. In the last few years, a great number of new drug delivery technologies have been optimised including the micro and nano-systems as well as polymer conjugation. Together, these drug delivery systems would not only improve drug administration and the efficiency and safety of conventional chemotherapies, but also revolutionize the pharmaceutical and biomedical industries in cancer therapy.

I. Introduction

The development of drug delivery systems has improved the therapeutic and toxicological properties of existing chemotherapies and facilitated the implementation of new ones. By including the drug in technologically optimized drug delivery systems or conjugating the drugs with different polymers, it is possible to modify the pharmacokinetics and biodistribution of the drugs, improving the efficacy and security of the therapy (Moses et al, 2003a).

Some of the strongest arguments for the use of drug delivery systems are that they avoid or at least reduce some potential disadvantages of cancer chemotherapy including toxicity, pain management, short in vivo half-lives and repeated administrations (Allen and Cullis, 2004). The possibility of designing different drug delivery systems for a controlled and continuous release of the therapeutic molecule has impact broadly the clinical application of the chemotherapies and improve the life-quality of the patients.

Currently, a variety of drug delivery approaches are FDA-approved or are in clinical development as anticancer treatments, including polymer microcapsules and microspheres, liposomes, polymer conjugates and nanoparticles (Figure 1). Others such as chemotherapy wafers, microchips and osmotic pumps are also in testing stage to treat human cancers but will not be the scope of this work since they have been reviewed in detail elsewhere (Moses et al, 2003b). This article focuses on the potential of micro and nanotechnology as well as polymer conjugation as a platform for developing drug delivery systems in cancer treatment.

II. Current limitations of chemotherapies

One of the main limitations of chemotherapies is their high toxicity which could lead to serious side effects, reducing the administrable and the therapeutic effect. To address this issue, it is essential to transport the therapeutically active molecule mainly to the target where

Figure 1. Examples of different drug delivery approaches that are FDA-approved or are in clinical development as anticancer treatments. Reproduced from Moses et al, 2003 with kind permission from Cancer Cell.

it is needed and at the required time and level (Orive et al, 2003a). This could be achieved by embedding the drugs into nontoxic and biodegradable polymers from which the drug will be released in a sustained manner (Duncan, 2003). For example, the use of doxorubicin in the treatment of metastatic breast cancer is associated with cardiotoxicity. But when the drug is included in a liposome, it is possible to obtain a controlled release of doxorubicin, reducing the peak levels of the drug and consequently its toxicity (Theodoulou and Clifford, 2004). Another strategy to concentrate cancer drugs only in their target tissue is through a mechanism known as enhanced permeability and retention effect (EPR) which happens in some pathological conditions such as solid tumors (Maeda et al, 2000; Hashizume et al, 2001). In fact, the network of blood vessels in many solid tumors has been shown to differ considerably from normal vasculature and to contain gaps in which tumor cells lack close contact with perfusing vessels, which ultimately leads to increased permeability (Jain, 2001; Bergers and Benjamin, 2003). In this situation, drug delivery systems which are usually excluded from entering into tissues can extravasate into tumors and increase drug concentration 10-fold or more than administration of the same dose of free drug (Northfelt et al, 1996).

In the last few years, many peptides and proteins have shown biological activity which makes them potential candidates as anticancer agents (Torchilin and Lukyanov, 2003). However, their use as therapeutic drugs is often hampered by the uptake by the reticulo endothelial system and their short half-lives in vivo which makes difficult their administration. The inclusion of a drug into a micro or nanoparticle or the coupling of a polymer decreases renal or hepatic drug clearance and immune recognition, altering the pharmacokinetics and biodistribution of the free drug. In fact, when a drug is associated with a carrier the volume of distribution and the clearance decreases, the area under time-versus-concentration increases and the half-life of the drug increases administrations (Allen and Cullis, 2004). This knowledge has provided the basis for different commercially available injectable delivery systems such as Decapeptyl^® and Zoladex^® (http://hcp.zoladex.net/Article/501611.aspx). Drug lifetime can also be prolonged by conjugating the drug molecules with water-soluble molecules such as polyethylene glycol (PEG) (Harris and Chess, 2003). The complex created is designed to increase protein solubility, stability and to reduce protein immunogenicity. In fact, by preventing rapid renal clearance and protein uptake of cells by reticuloendothelial system, conjugates with PEG are used to prolong plasma half-time (Gabizon et al, 2003; Shorr et al, 2004).

The use of drug delivery systems have revolutionised the areas of cancer prevention and pain management related to classical cancer chemotherapy. Furthermore, the encapsulation of the drug in a microreservoir can improve drug solubility and stability as well as reduce drug resistance in some human carcinomas. For instance, recently it has been demonstrated that liposomally encapsulated tamoxifen not only induces pharmacological effects but reduces anti-estrogen resistance in several breast tumour models (Reiner et al, 2004). Finally, the entrapment of labile therapeutic agents can protect the latter from premature degradation by hydrolysis or by enzymes present in the plasma.

III. Micro and nanotechnologies

Tremendous opportunities exist for using micro and nanoparticles as controlled drug delivery systems for cancer treatment (Panyam and Labhasetwar, 2003; Birnbaum and Brannon-Peppas, 2004). The term “microparticle” refers to a particle with a diameter of 1-1000 mm, while “nanopaticle” is used when the particle is <1 mm in size. However, under this term it is possible to distinguish several reservoirs including micro/nano-capsules, micro/nano-spheres, liposomes, etc. All these devices differ not only in the structure (Figure 2) but also in their biopharmaceutical properties and therapeutic uses (Orive et al, 2003b). The fabrication protocol of each particle differs also considerably and the scale-up could be a challenge for some of these devices.

An important issue to be considered when fabricating these systems is the drug load that the reservoir can carry. This drug load depends on the size and the structure of the device, ranging from some few molecules of the drug to a few tens. Therefore, selection of drugs with potent pharmaceutical activity is necessary in order to have therapeutic effects in the released dose. Furthermore, it is essential that the drug will not be altered during the fabrication process and the storage. Finally, interactions between drug and the reservoir must be optimized to facilitate drug release only in the target where it is needed and at the desired kinetic-release.

Natural and synthetic polymers including albumin, fibrinogen, alginate, chitosan and collagen have been used for the fabrication of micro and nanoparticles . However, among all of them, lactic-glycolic acid copolymers are the most frequently employed materials due to their biocompatibility and biodegradability. Following a multiple emulsion process, a drug can be entrapped into a poly(lactic-co-glycolic) (PLGA) microsphere and released

Figure 2. Schematics of different nanotechnology based drug delivery systems for cancer therapy. Reproduced from Sahoo and Labhasetwar, 2003 with kind permission from Drug Discovery Today.

at a zero-order kinetic by diffusion of the drug through the polymer reservoir and the slow degradation of the polymer matrix. These advantages resulted in the first two PLGA-microparticle extended-release formulations that were approved by the US Food and Drug Administration (FDA). One of them released the recombinant human growth hormone (rhGH) (Cleland, 1997) whereas the other microparticle-based drug delivery system released the leuteinizing-hormone-releasing hormone (LHRH) agonist leuprorelin acetate (Okada, 1997). The latter is currently on the market under the name of Lupron^® Depot and it is approved in the United States for the palliative treatment of advanced prostate cancer. However, there are still few microencapsulated formulations on clinical trials addressing cancer treatments. In fact, a recent review of National Centre Institute revealed that from the 1200 open clinical trials in the United States only one to be testing a microparticulate system for controlled drug delivery (Birnbaum and Brannon-Peppas, 2004). Experts, however, predict that within the next 5-10 years some of the formulations currently under study might progress to the clinical evaluation and perhaps become marketed therapy not so far (http://www.zycos.com/press/release15.html; Hedley et al, 1998).

Nanotechnology is a multidisciplinary field which encompasses research and development at the atomic, molecular or macromolecular level (Emerich and Thanos, 2003). Due to their extremely small size, nanoscale structures have unique properties for the controlled and targeted release of therapeutic products (Sahoo and Labhasetwar, 2003). In the last few years, investment in nanotechnology worldwide has increased considerably and recently the National Cancer Institute has announced a major commitment to nanotechnology for cancer research in the form of $144.3 million, five-year initiative.

Although the total drug-load is reduced considerably and the manufacture process is more complex, the nanoscale devices present some advantages over the micro-systems. In fact, submicron systems show higher intracellular uptake than microsized particles, thereby allowing drug-release in different cellular compartments such as cytoplasm and nucleus. Nanoparticles can be also easily conjugated with a ligand to favour a targeted therapeutic approach and as it has been reported, some nanoparticles can cross the blood-brain barrier (BBB). For example, doxorubicin bound to polysorbate-coated nanoparticles can cross the intact BBB, reaching therapeutic concentrations in the brain. When these particles were administered in glioblastoma-bearing rats, a very aggressive human cancer with short survival times, significantly higher survival times were observed in the treated animal group compared with all other groups (Steiniger et al, 2004). Depending on the elaboration method and the materials employed different nano-systems can be distinguished including micelles, nanocapsules, dendrimers, nanospheres, solid lipid nanoparticles and ceramic nanoparticles. The principal characteristics and some of the recent research using each nano-systems is reviewed in Table 1.

Liposomes are one of the most well-known drug delivery carriers employed in the treatment of cancer. Due to their advantages, liposomal formulations provide a substantial increase in antitumor efficacy comparing with the free drug or standard chemotherapy regimens (Drummond et al, 2004). Liposomes are composed of a double lipid bilayer which encloses an aqueous space that can be employed to transport anticancer drugs.

Table 1. Examples of different nanoparticles and their applications as cancer treatments

Nanoparticle	Description	Recent applications	Reference
Nanocapsules	Vesicular systems in which the drug is surrounded by a polymeric membrane	Stability of the cisplatin nanocapsules has been optimized by varying the lipid composition of the bilayer coat	Velinova, 2004
Nanospheres	Matrix systems in which the drug is physically and uniformly dispersed	Bovine serum albumin nanospheres containing 5-fluorouracil show higher tumour inhibition than the free drug	Santhi, 2002
Micelles	Amphiphilic block copolymers that can self-associate in aqueous solution	Micelle delivery of doxorubicin increases cytotoxicity to prostate carcinoma cells	McNaealy, 2004
Ceramic nanoparticles	Nanoparticles fabricated using inorganic compounds including silica, titania…	Ultra fine silica based nanoparticles releasing water insoluble anticancer drug	Roy, 2003
Liposomes	Artificial spherical vesicles produced from natural phospholipids and cholesterol	Radiation-guided drug delivery of liposomal cisplatin to tumor blood vessels results in improved tumour growth delay	Geng, 2004
Dendrimers	Macromolecular compound that comprise a series of branches around an inner core	Targeted delivery within dendrimers improved the cytotoxic response of the cells to methotrexate 100-fold over free drug	Quintana, 2002
SLN particles	Nanoparticles made from solid lipids	SLN powder formulation of all-trans retinoic acid may have potential in cancer chemoprevention and therapeutics.	Soo-Jeong, 2004

Some factors must be taken into account when preparing the liposomal formulations including the size, the surface charge (Senior, 1987) and the membrane fluidity (Gregoriadis and Senior, 1980). All these formulation issues have implications on the pharmacokinetics, biodistribution and bioavailability of the entrapped therapeutic product.

There are many liposomes for the treatment of malignancies that are already approved, awaiting approval or in clinical trials. The most frequently studied drug family is the anthracyclines since they present activity against a wide range of tumours (Young et al, 1981). For example, doxorubicin liposomes have shown significant activity against AIDS-related KaposiΪs sarcoma, breast and ovarian cancers in different clinical trials (Amantea et al, 1997; Muggia, 1997; Ranson et al, 1997). Liposomes containing daunorubicin have been also successfully employed in the treatment of KaposiΪs sarcoma and are currently currently being evaluated with some effectiveness for the treatment of central nervous system tumors (Zucchetti et al, 1999). Another promising liposomal product is the vincristine liposome. In fact, in a preliminary phase II trial for the treatment of non-HodginΪs lymphomas, liposomal-vincristine showed efficacy against the transformed or aggressive non-HodginΪs lymphomas and presented less neurotoxicity than the free-drug (Sarris et al, 2000).

Another interesting approach is the immobilization of therapeutic product-secreting cells within microcapsules (Orive et al, 2003c, 2004). Enclosing the biologically active material within a polymeric matrix surrounded by a semipermeable membrane it is possible to circumvent immune rejection while enabling the controlled and continuous release of the active substance. Several attemps of applying this technology to treatment of different malignancies have been described and reviewed in the literature (Orive et al, 2003d). For instance, using CYP2B1-transfected cells which activate the prodrug ifosfamide at the site of tumor, the median survival of mice transplanted with a human pancreatic carcinoma was the double than the control group (Lörh et al, 2002). The safety of this protocol was evaluated in a phase I/II trial in 14 patients with pancreatic cancer. Results showed that the tumours of four patients regressed after treatment and those of other ten individuals who followed the study remain stable (Lörh et al, 2001).

However, the inhibition of tumour angiogenesis has been so far the most frequently targeted phenomenon by microencapsulated cells. Angiogenesis is the process by which new blood vessels are formed to promote tumour growth and metastasis (Folkman, 1971). The available evidence suggest that optimal anti-angiogenic therapy requires prolonged exposure to low drug concentrations (Cristofanalli et al, 2002; Kerbel and Folkman, 2002; Sweeney et al, 2003). Furthermore, the discovery of endogenous antiangiogenic factors (OΪReally et al, 1994, 1997) has opened the door to the genetic manipulation of cells and consequently their encapsulation in polymer matrices (Boüard et al, 2003; Cirone et al, 2003). Using this strategy, genetically engineered kidney epithelial cells expressing the anti-angiogenic agent endostatin were assayed for the treatment of glioblastomas (Joki et al, 2001). Mice inoculated with human glioma U87MG cells were divided in three groups. The first group received a single injection of microcapsules containing endostatin secreting cells (BHK-endo). The second received an injection of neomycin-resistant gene-transfected cells that did not secrete endostatin (BHK-neo) and the third group did not receive any treatment (control). Results showed that in comparison with controls, the growth of glioma tumours was suppressed by 62% by 21 days post administration of the encapsulated BHK-neo cells (Figure 3A). Furthermore, the BHK-endo group showed a 72% reduction in tumour weight when compared to the other two groups (Figure 3B) (Joki et al, 2001). A similar approach done by other research group resulted in analogous results with a tumor growth reduction of >70% (Read et al, 2001). In addition, encapsulated cells secreting angiostatin have been combined with immunotherapy against a mouse melanoma model showing improved survival with 30% of the animals surviving tumour free (Cirone et al, 2004).

IV. Macromolecular conjugation

The pharmacokinetics and biodistribution of the cancer drugs can be improved by conjugating the latter with water soluble polymers, peptides and proteins. PEG is the main representative of the polymers used to conjugate with the therapeutic drug in order to improve the pharmacokinetics of the latter. In fact, since the first PEGylated protein (PEG-adenosine deaminase) entered the market in 1990 (Levy et al, 1988), a large number PEGylated pharmaceuticals have followed. For instance, PEG-interferon a-2b is under clinical evaluation in a phase II randomized study in patients with metastatic or unresectable carcinoid tumours whereas PEGylated-L-asparaginase (Oncospar^®) is used in the treatment of acute leukaemia. The latter can be administered every 2 weeks instead of the 2-3 times per week needed for the native enzyme. In addition, a Phase II clinical trial is undergoing to evaluate PEGylated-L-asparaginase in multiple myeloma (http://www.clevelandclinic.org/myeloma /pegasp.htm. This strategy can be also used to improve targeting and EPR effect of polymer particles. In fact, by conjugating specific vector molecules to micro and nanoparticles it is possible to improve the affinity towards different tissues or tumours. Targeted therapy enables a higher bioavailability of the therapeutic molecules whereas reduces considerably possible side-effects. Vector molecules capable of recognizing tumors include antibodies, lectins, peptides, hormones, folate and vitamins. For example, monoclonal nuclear antibodies can be attached to the drug carriers to promote drug release only to tumour cells and not normal cells (Iakoubov et al, 1995). Interestingly, the high affinity of folic acid for folate receptors provides a unique opportunity to use folic acid as a targeting ligand to deliver chemotherapeutic agents to cancer cells. In vitro experiments using folate-tethered liposomes containing calcein or doxorubicin showed a selective drug release in both human cervical cancer HeLa-IU1 cells and human colon cancer Caco-2 cells overexpressing folate receptors (Zhang et al, 2004)

Figure 3. The effect of encapsulated endostating secreting cells (BHK-endo), encapsulated cells that did not secrete endostatin (BHK-neo) and no treatment (control) on tumor growth in vivo. A) Tumour size and B) tumour weight of subcutaneous U87MG human glioma cell xenograft. Reproduced from Joki et al, 2001 with kind permission from Nature Biotechnololy.

In another approach, Hasokawa et al. used a human monoclonal antibody formulated as PEG-modified immunoliposomal doxorubicin to treat selectively human stomach cancer. In vivo experiments showed greater antitumor activity than the unmodified liposome or the free drug (Hosokawa et al, 2003). Anti-tumor antibody-conjugated polymeric micelles called immunomicelles have shown successful in vitro and in vivo delivery of taxol into various cancer cells (Torchilin et al, 2003). In fact, when inhibition of murine Lewis lung carcinoma tumour growth in mice was evaluated with different taxol preparations, the average weight of excised tumours in the group treated with taxol incorporated in immunomicelles was 0.67±0.35 g compared with 1.58±0.48 g and 1.37±0.36 g in groups treated with free taxol or taxol in plain PEG-PE micelles, respectively (P < 0.05 in both cases) (Figure 4).

Figure 4. Inhibition of murine Lewis lung carcinoma tumour growth in mice with different taxol preparations including free taxol, plain micelles and taxol releasing 2C5-immunomicelles. Reproduced from Torchilin et al, 2003 with kind permission from Proc. Natl. Acad. Sci. U.S.A.

Finally, modified dendrimers have been also fully investigated for targeted drug delivery. Dendrimers associated with an anionic oligomer were elaborated for delivering angiostatin and tissue inhibitor of metalloproteinase (TIMP)-2. In vivo results showed that angiostatin release inhibited tumour growth by 71% while (TIMP)-2 by 84%. Moreover, combined therapy resulted in 96% inhibition of tumour growth (Vincent et al, 2003).

V. Conclusions

The present paper reviews the use of micro and nanotechnology as well as macromolecular conjugation as strategies to deliver existing chemotherapies and novel therapeutic molecules in a controlled manner to malignancies. These technologies come along with other exciting drug delivery approaches such as patches, microchips and osmotic pumps. In general, the technologies described here improve significantly the pharmacokinetics and biodistribution of the free drugs and reduce considerably their side-effects. Furthermore, some of them have been approved by FDA and are currently in the market.

In the future, some challenges need to be addressed including the adaptation of each drug delivery system to the particular needs of each malignancy, improvement of interactions between the drug and some of the components of the carrier and development of multifunctional systems able to deliver several drugs at the same or different time with different kinetic releases.

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