Oligonucleotide delivery in cancer therapy

Importance of the field: Cancer is frequently caused by altered protein expression. Oligonucleotides (ONs) are short synthetic nucleic acid fragments, able to selectively correct protein expression into cells by different mechanisms. However, biological barriers hamper the therapeutic use of ONs without suitable delivery strategies.

Areas covered in this review: This review summarizes the most meaningful non-viral strategies for ON delivery, including the chemical modifications of the ON backbone and non-viral delivery systems.

What the reader will gain: The reader will gain an update of the main strategies for ON delivery in cancer. Advantages and limits of each approach are underlined. Emphasis is given to the delivery strategies that contributed to bringing ONs into clinical trials.

Take home message: In the long story of ONs for cancer therapy, the development of delivery strategies has led, in the last few years, to different opportunities to use the high therapeutic potential of these molecules in humans.     

Recent advances in the targeting of systemically administered non-viral gene delivery systems

ABSTRACT

Introduction: Systemically administered non-viral gene delivery systems face multiple biological barriers that decrease their efficiency. These systems are rapidly cleared from the circulation and sufficient concentrations do not accumulate in diseased tissues. A number of targeting strategies can be used to provide for sufficient accumulation in the desired tissues to achieve a therapeutic effect.

Areas covered: We discuss recent advances in the targeting of non-viral gene delivery systems to different tissues after systemic administration. We compare passive and active targeting applied for tumor delivery and propose some strategies that can be used to overcome the drawbacks of each case. We also discuss targeting the liver and lungs as two particularly important organs in gene therapy.

Expert opinion: There is currently no optimum non-viral gene delivery system for targeting genes to specific tissues. The dose delivered to tumor tissues using passive targeting is low and shows a high patient variation. Although active targeting can enhance binding to specific cells, only a few reports are available to support its value in vivo. The design of smart nanocarriers for promoting active targeting is urgently needed and targeting the endothelium is a promising strategy for gene delivery to tumors as well as other organs.     

Ultrasound-mediated gene delivery

Importance of the field: The use of ultrasound with microbubbles raises the possibility of an efficient and safe gene delivery.

Areas covered in this review: This review summarizes the current state of the art of gene delivery by sonoporation under the following topics. First, the basic ultrasound parameters and the characteristics of microbubble in biological systems are discussed. Second, the extensions of sonoporation to other fields of gene delivery such as viral and non-viral vector are briefly reviewed. Finally, recent applications in an animal model for various diseases are introduced.

What the reader will gain: Information and comments on gene delivery by sonoporation or enhanced cell membrane permeability by means of ultrasound.

Take home message: Ultrasound-mediated gene delivery combined with microbubble agents provides significant safety advantages over other methods of local gene delivery.     

Oral delivery of peptides and proteins using lipid-based drug delivery systems

Introduction: In order to successfully develop lipid-based drug delivery systems (DDS) for oral administration of peptides and proteins, it is important to gain an understanding of the colloid structures formed by these DDS, the mode of peptide and protein incorporation as well as the mechanism by which intestinal absorption of peptides and proteins is promoted.

Areas covered: The present paper reviews the literature on lipid-based DDS, employed for oral delivery of peptides and proteins and highlights the mechanisms by which the different lipid-based carriers are expected to overcome the two most important barriers (extensive enzymatic degradation and poor transmucosal permeability). This paper also gives a clear-cut idea about advantages and drawbacks of using different lipidic colloidal carriers ((micro)emulsions, solid lipid core particles and liposomes) for oral delivery of peptides and proteins.

Expert opinion: Lipid-based DDS are safe and suitable for oral delivery of peptides and proteins. Significant progress has been made in this area with several technologies on clinical trials. However, a better understanding of the mechanism of action in vivo is needed in order to improve the design and development of lipid-based DDS with the desired bioavailability and therapeutic profile.     

Poly(amidoamine) dendrimer complexes as a platform for gene delivery

Introduction: Gene therapy is one of the most effective ways to treat major infectious diseases, cancer and genetic disorders. It is based on several viral and non-viral systems for nucleic acid delivery. The number of clinical trials based on application of non-viral drug and gene delivery systems is rapidly increasing.

Areas covered: This review discusses and summarizes recent advances in poly(amidoamine) dendrimers as effective gene carriers in vitro and in vivo, and their advantages and disadvantages relative to viral vectors and other non-viral systems (liposomes, linear polymers) are considered.

Expert opinion: In this regard, dendrimers are non-immunogenic and have the highest efficiency of transfection among other non-viral systems, and none of the drawbacks characteristic for viral systems. The toxicity of dendrimers both in vitro and in vivo is an important question that has been addressed on many occasions. Several non-toxic and efficient multifunctional dendrimer-based conjugates for gene delivery, along with modifications to improve transfection efficiency while decreasing cytotoxicity, are discussed. Twelve paradigms that affected the development of dendrimer-based gene delivery are described. The conclusion is that dendrimers are promising candidates for gene delivery, but this is just the beginning and further studies are required before using them in human gene therapy.     

Spotlight on the delivery of photosensitizers: different approaches for photodynamic-based therapies

Introduction: Nanomedicine development allowed the discovery of new photosensitizers (PS) and drug delivery systems (DDS) to overcome current issues on phototherapy. Nano-engineered materials have the potential to improve the solubility of PS, control drug pharmacokinetics, decreasing side effects, increasing bioavailability, and overcoming multidrug resistance. A recent approach is the co-delivery of PS with other therapeutic agents in a multimodal platform for synergic and improved results.

Areas covered: This paper discusses the delivery of PS-nanostructured platforms for conventional, photothermal, and antimicrobial photodynamic therapies, as well as in a recent therapeutic modality for photobiomodulation, covering applications of cancer diagnosis, targeting to skin pathogens, photoregeneration and wound healing. The focus of the present review is to describe the use of different DDS to enhance the therapeutic outcomes triggered by the combination of delivered PS, light, and oxygen.

Expert opinion: Nanotechnology allowed the development of site-specific delivery of PS molecules, expanding possibilities poorly explored before to enhance photodynamic efficacy and extrapolate the concept to other treatment protocols. Research in this area embraces potential and pitfalls of PS delivery, allowing new clinical phase outcomes and long-term issues to be established, which will impact on several biomedical applications.     

Applications of Hemagglutinating Virus of Japan in therapeutic delivery systems

Background: Efficient and minimally invasive vector systems appear to be the most appropriate for both gene therapy and drug delivery. Numerous viral and non-viral vectors have been developed. Each vector has its own advantages and limitations. Objective: New vectors have been required for overcoming the limitations of both viral vectors and non-viral vectors. The idea is to compensate the limitations of one vector system with the advantages of another. This can enable efficient drug delivery and gene expression, while reducing the cytotoxicity of the various vector components. Methods: The Hemagglutinating Virus of Japan (HVJ; Sendai virus) envelope vector was developed using fusion-competent inactivated HVJ particle. Briefly, the viral genome was destroyed by UV-irradiation, and the inactivated viral particles were mixed with plasmid DNA, proteins or siRNA in the presence of mild detergent. After centrifugation, those molecules were incorporated into the viral envelope. Conclusion: The HVJ-E vector can efficiently deliver therapeutic molecules such as genes, siRNA, decoy oligonucleotides, proteins, and anti-cancer drugs to various tissues in vivo. It is also available for high throughput screening of therapeutic genes. A number of anti-cancer effects of HVJ-E have been identified, specifically activation of both T cell immunity and non-T cell immunity against cancers. Furthermore, a tissue-targeting HVJ-E vector has been constructed using a unique approach for virus engineering and by conjugation with biocompatible polymers. Therefore, the HVJ-E vector is expected to enable effective cancer therapy through the delivery of molecular therapy and through its immunotherapeutic effects.     

Endosomal disruptors in non-viral gene delivery

Importance of the field: Non-viral gene delivery for the treatment of genetic and non-genetic diseases has been under investigation for several decades, but there has been very little application in patients because of poor gene expression and toxicity.

Areas covered in this review: As gene delivery almost invariably involves endocytosis, many of its limitations are related to compartmentalisation of the transgene within the endosomes. Gene expression enhancers have become an essential part of manipulating endosomal release, as well as protecting transgene from intracellular degradation. However, disruption of the endosomes can also release proteases that have been shown to activate apoptotic pathways.

What the reader will gain: An understanding of the role that endosomal release plays in the toxicity of gene delivery vehicles will help identify new approaches to minimise adverse effects while enhancing non-viral gene expression.

Take home message: The future of non-viral gene therapy needs to identify new approaches that limit endosome-induced toxicity while enhancing expression so that a pharmacological response can be reliably observed in vivo.     

Targeted delivery of etoposide to cancer cells by folate-modified nanostructured lipid drug delivery system

Abstract

Context: Cardiotoxicity and myelosuppression of etoposide (ETP) limited its clinical application. Targeted drug delivery system could deliver anticancer agents to the target cancerous cells, thus reducing their toxicity.

Objective: In this study, folate (FA) was applied for the construction of nanostructured lipid carriers (NLCs), and used for targeted delivery of ETP to tumors overexpresses the FA receptors.

Methods: FA-poly (ethylene glycol)-distearoylphosphatidylethanolamine was synthesized. FA decorated and ETP-loaded NLCs (FA-ETP-NLCs) were prepared and the formulation was optimized by Box–Behnken design. Their particle size (PS), zeta potential and drug encapsulation efficiency (EE) was evaluated. In vitro cytotoxicity studies of FA-ETP-NLCs were tested in CT26, SGC7901, NCI-H209 cell lines. In vivo antitumor efficacies of the carriers were evaluated on mice bearing CT26 cells xenografts.

Results: The optimum FA-ETP-NLCs formulations had a PS of 120.86?nm. The growth of CT26, SGC790 or NCI-H209 cells in vitro was obviously inhibited. FA-ETP-NLCs also displayed the best antitumor activity than other formulations in vivo.

Conclusion: The results demonstrated that FA-ETP-NLCs were efficient in selective delivery to CT26, SGC790 or NCI-H209 cells overexpressing the FA receptors. Also, FA-ETP-NLCs can sufficiently transfer ETP to the cancer cells, enhance the antitumor capacity. Thus, FA-ETP-NLCs could prove to be a superior nanomedicine to achieve tumor therapeutic efficacy.     

Cyclodextrin-based siRNA delivery nanocarriers: a state-of-the-art review

Introduction: The discovery of synthetic small interfering RNA (siRNA) has led to a surge of interest in harnessing RNA interference (RNAi) technology for biomedical applications and drug development. Even though siRNA can be a powerful therapeutic drug, its delivery remains a major challenge, due to the difficulty in its cellular uptake. Naked siRNA has a biological half-life of less than an hour in human plasma. To increase the lifetime and improve its therapeutic efficacy, non-viral vectors have been developed. As a natural evolution, cyclodextrins (CDs), which are natural cyclic oligosaccharides, have recently been applied as delivery vehicles for siRNA, and this in turn, has led to a surge of interest in this area.

Areas covered: This review discusses the recent advances made in the design of delivery strategies for siRNA, focusing on CD-based delivery vectors, because these have demonstrated clinical success. The methods of preparation of CD-based vectors, their characterization, transfection efficiencies, cellular toxicity, preclinical and clinical trials are also addressed, as well as future therapeutic applications.

Expert opinion: siRNA-mediated RNAi therapeutics is beginning to transform healthcare, particularly, for the treatment of solid tumors. For example, CALAA01, a targeted, self-assembling nanoparticle system based on CD complexed with siRNA has been effective in phase I clinical trials. Although siRNA therapeutics suffers from problems related to off-target effects and non-specific gene silencing, these problems can be overcome by reducing the nanoparticle size, improving the targeting efficiency and by modifying the primary sequence of the siRNA.     

Therapeutic applications of hydrogels in oral drug delivery

Introduction: Oral delivery of therapeutics, particularly protein-based pharmaceutics, is of great interest for safe and controlled drug delivery for patients. Hydrogels offer excellent potential as oral therapeutic systems due to inherent biocompatibility, diversity of both natural and synthetic material options and tunable properties. In particular, stimuli-responsive hydrogels exploit physiological changes along the intestinal tract to achieve site-specific, controlled release of protein, peptide and chemotherapeutic molecules for both local and systemic treatment applications.

Areas covered: This review provides a wide perspective on the therapeutic use of hydrogels in oral delivery systems. General features and advantages of hydrogels are addressed, with more considerable focus on stimuli-responsive systems that respond to pH or enzymatic changes in the gastrointestinal environment to achieve controlled drug release. Specific examples of therapeutics are given. Last, in vitro and in vivo methods to evaluate hydrogel performance are discussed.

Expert opinion: Hydrogels are excellent candidates for oral drug delivery, due to the number of adaptable parameters that enable controlled delivery of diverse therapeutic molecules. However, further work is required to more accurately simulate physiological conditions and enhance performance, which is important to achieve improved bioavailability and increase commercial interest.     

Serum influence on in-vitro gene delivery using microbubble-assisted ultrasound

Abstract

Background: Plasmid DNA (pDNA) is attractive molecule for gene therapy. pDNA-targeted delivery by efficient and safe methods is required to enhance its intra-tissue bioavailability. Among non-viral methods, sonoporation has become a promising method for in-vitro and in-vivo pDNA delivery. The efficiency of non-viral delivery methods of pDNA is generally limited by the presence of serum.

Purpose: The aim of this study was to evaluate the influence of serum on in-vitro pDNA delivery using microbubble-assisted ultrasound.

Methods: The effects of a range of serum concentrations (0–50%) on efficiency of in-vitro pDNA delivery by sonoporation were determined on human glioblastoma cells. Furthermore, the influence of the serum on cell viability, membrane permeabilization, microbubble destruction, and pDNA topology were also assessed.

Results: In-vitro results showed that a low serum concentration (i.e. ≤1%) induced a significant increase in transfection level through an increase in cell viability. However, a high serum concentration (i.e. ≥5%) resulted in a significant decrease in cell transfection, which was not associated with a decrease in membrane permeabilization or loss in cell viability. This decrease in transfection level was in fact positively correlated to changes in pDNA topology.

Conclusion: Serum influences the efficiency of in-vitro pDNA delivery by sonoporation through change in pDNA topology.     

Biodegradable polymers: an update on drug delivery in bone and cartilage diseases

ABSTRACT

Introduction: The unique structure of bone and cartilage makes the systemic delivery of free drugs to those connective tissues very challenging. Consequently, effective and targeted delivery for bone and cartilage is of utmost importance. Engineered biodegradable polymers enable designing carriers for a targeted and temporal controlled release of one or more drugs in concentrations within the therapeutic range. Also, tissue engineering strategies can allow drug delivery to advantageously promote the in situ tissue repair.

Areas covered: This review article highlights various drug delivery systems (DDS) based on biodegradable biomaterials to treat bone and/or cartilage diseases. We will review their applications in osteoporosis, inflammatory arthritis (namely osteoarthritis and rheumatoid arthritis), cancer and bone and cartilage tissue engineering.

Expert opinion: The increased knowledge about biomaterials science and of the pathophysiology of diseases, biomarkers, and targets as well as the development of innovative tools has led to the design of high value-added DDS. However, some challenges persist and are mainly related to an appropriate residence time and a controlled and sustained release over a prolonged period of time of the therapeutic agents. Additionally, the poor prediction value of some preclinical animal models hinders the translation of many formulations into the clinical practice.     

Recent advances of siRNA delivery by nanoparticles

Introduction: The field of RNA interference technology has been researched extensively in recent years. However, the development of clinically suitable, safe and effective drug delivery vehicles is still required.

Areas covered: This paper reviews the recent advances of non-viral delivery of small interfering RNA (siRNA) by nanoparticles, including biodegradable nanoparticles, liposomes, polyplex, lipoplex and dendrimers. The characteristics, composition, preparation, applications and advantages of different nanoparticle delivery strategies are also discussed in detail, along with the recent progress of non-viral nanoparticle carrier systems for siRNA delivery in preclinical and clinical studies.

Expert opinion: Non-viral carrier systems, especially nanoparticles, have been investigated extensively for siRNA delivery, and may be utilized in clinical applications in the future. So far, a few preliminary clinical trials of nanoparticles have produced promising results. However, further research is still required to pave the way to successful clinical applications. The most important issues that need to be focused on include encapsulation efficiency, formulation stability of siRNA, degradation in circulation, endosomal escape and delivery efficiency, targeting, toxicity and off-target effects. Pharmacology and pharmacokinetic studies also present another great challenge for nanoparticle delivery systems, owing to the unique nature of siRNA oligonucleotides compared with small molecules.     

Current status of non-viral gene therapy for CNS disorders

ABSTRACT

Introduction: Viral and non-viral vectors have been used as methods of delivery in gene therapy for many CNS diseases. Currently, viral vectors such as adeno-associated viruses (AAV), retroviruses, lentiviruses, adenoviruses and herpes simplex viruses (HHV) are being used as successful vectors in gene therapy at clinical trial levels. However, many disadvantages have risen from their usage. Non-viral vectors like cationic polymers, cationic lipids, engineered polymers, nanoparticles, and naked DNA offer a much safer option and can therefore be explored for therapeutic purposes.

Areas covered: This review discusses different types of viral and non-viral vectors for gene therapy and explores clinical trials for CNS diseases that have used these types of vectors for gene delivery. Highlights include non-viral gene delivery and its challenges, possible strategies to improve transfection, regulatory issues concerning vector usage, and future prospects for clinical applications.

Expert opinion: Transfection efficiency of cationic lipids and polymers can be improved through manipulation of molecules used. Efficacy of cationic lipids is dependent on cationic charge, saturation levels, and stability of linkers. Factors determining efficacy of cationic polymers are total charge density, molecular weights, and complexity of molecule. All of the above mentioned parameters must be taken care for efficient gene delivery.     

Specific delivery of kinase inhibitors in nonmalignant and malignant diseases

Introduction: Kinase inhibitors have been hailed as a breakthrough in the treatment of cancer. Extensive research is now being devoted to the development of kinase inhibitors as a treatment for many nonmalignant diseases. However, the use of kinase inhibitors in both malignant and nonmalignant diseases is also associated with side effects and the development of resistance. It may be worthwhile to explore whether cell-specific delivery of kinase inhibitors improves therapeutic efficacy and reduces side effects.

Areas covered: This review aims to provide an overview of the preclinical studies performed to examine the specific targeting of kinase inhibitors in vitro and in vivo. It gives an introduction to kinase signaling pathways induced during disease, along with the possible problems associated with their inhibition. It also discusses the studies on specific delivery and shows that altering the specificity of kinase inhibitors by targeting methods improves their effectivity and safety.

Expert opinion: Compared with the delivery of cytotoxic compounds, the specific delivery of kinase inhibitors has not yet been studied extensively. The studies discussed in this review provide an insight into methods used to target kinase inhibitors to different organs. The targeting of different kinase inhibitors has improved their therapeutic possibilities, but many questions still remain to be studied.     

P53-Derived peptides conjugation to PEI: an approach to producing versatile and highly efficient targeted gene delivery carriers into cancer cells

ABSTRACT

Objectives: Targeted delivery of cytotoxic drugs or therapeutic antisense RNAs into specific cells is a major bottleneck in cancer therapy. To overcome this problem and improve the specificity for cancer cells, we describe a new-targeted delivery system using p53-derived peptides, namely PNC 27 and PNC 28. These peptides target HDM-2 on the surface of cancer cells. HDM-2 is overexpressed on the surface of cancerous cells, but not present on the untransformed cells.

Methods: To determine HDM-2-expressing cells, we used immunocytochemistry and flow cytometry analysis on nine cell lines including MCF-7 and NIH-3t3. Conjugation of peptides to vectors was confirmed using reverse-phase high-pressure liquid chromatography (RP-HPLC). Physicochemical properties of vector/DNA complexes including particle size, surface charge and DNA condensation ability were determined. In transfection studies, three plasmids were used including luciferase, pEGFP and shRNA plasmid against Bcl-XL mRNA. The level of Bcl-XL expression was determined by real-time PCR and western blot techniques.

Results: The results of gene delivery and shRNA-based gene silencing studies indicated that conjugation of PNC peptides could enhance gene delivery efficiently with high-targeted activity exclusively into cancer cells.

Conclusion: Our results strongly indicated that this targeting system could be utilized as an efficient targeting method for most cancer cells.     

Strategies on the nuclear-targeted delivery of genes

Abstract

To improve the nuclear-targeted delivery of non-viral vectors, extensive effort has been carried out on the development of smart vectors which could overcome multiple barriers. The nuclear envelope presents a major barrier to transgene delivery. Viruses are capable of crossing the nuclear envelope to efficiently deliver their genome into the nucleus through the specialized protein components. However, non-viral vectors are preferred over viral ones because of the safety concerns associated with the latter. Non-viral delivery systems have been designed to include various types of components to enable nuclear translocation at the periphery of the nucleus. This review summarizes the progress of research regarding nuclear transport mechanisms. “Smart” non-viral vectors that have been modified by peptides and other small molecules are able to facilitate the nuclear translocation and enhance the efficacy of gene expression. The resulting technology may also enhance delivery of other macromolecules to the nucleus.     

Cell-penetrating peptides for the delivery of nucleic acids

Introduction: Different gene therapy approaches have gained extensive interest lately and, after many initial hurdles, several promising approaches have reached to the clinics. Successful implementation of gene therapy is heavily relying on finding efficient measures to deliver genetic material to cells. Recently, non-viral delivery of nucleic acids and their analogs has gained significant interest. Among non-viral vectors, cell-penetrating peptides (CPPs) have been extensively used for the delivery of nucleic acids both in vitro and in vivo.

Areas covered: In this review we will discuss recent advances of CPP-mediated delivery of nucleic acid-based cargo, concentrating on the delivery of plasmid DNA, splice-correcting ONs, and small-interfering RNAs.

Expert opinion: CPPs have proved their potential as carriers for nucleic acids. However, similarly to other non-viral vectors, CPPs require further development, as efficient systemic delivery is still seldom achieved. To achieve this, CPPs should be modified with entities that would allow better endosomal escape, targeting of specific tissues and cells, and shielding agents that increase the half-life of the vehicles. Finally, to understand the clinical potential of CPPs, they require more thorough investigations in clinically relevant disease models and in pre-clinical and clinical studies.     

Low-frequency sonophoresis: application to the transdermal delivery of macromolecules and hydrophilic drugs

Importance of the field: Transdermal delivery of macromolecules provides an attractive alternative route of drug administration when compared to oral delivery and hypodermic injection because of its ability to bypass the harsh gastrointestinal tract and deliver therapeutics non-invasively. However, the barrier properties of the skin only allow small, hydrophobic permeants to traverse the skin passively, greatly limiting the number of molecules that can be delivered via this route. The use of low-frequency ultrasound for the transdermal delivery of drugs, referred to as low-frequency sonophoresis (LFS), has been shown to increase skin permeability to a wide range of therapeutic compounds, including both hydrophilic molecules and macromolecules. Recent research has demonstrated the feasibility of delivering proteins, hormones, vaccines, liposomes and other nanoparticles through LFS-treated skin. In vivo studies have also established that LFS can act as a physical immunization adjuvant. LFS technology is already clinically available for use with topical anesthetics, with other technologies currently under investigation.

Areas covered in this review: This review provides an overview of mechanisms associated with LFS-mediated transdermal delivery, followed by an in-depth discussion of the current applications of LFS technology for the delivery of hydrophilic drugs and macromolecules, including its use in clinical applications.

What the reader will gain: The reader will gain an insight into the field of LFS-mediated transdermal drug delivery, including how the use of this technology can improve on more traditional drug delivery methods.

Take home message: Ultrasound technology has the potential to impact many more transdermal delivery platforms in the future due to its unique ability to enhance skin permeability in a controlled manner.