Progress and Outlook of Inorganic Nanoparticles for Delivery of Nucleic Acid Sequences Related to Orthopedic Pathologies: A Review

Abstract

The anticipated growth in the aging population will drastically increase medical needs of society; of which, one of the largest components will undoubtedly be from orthopedic-related pathologies. There are several proposed solutions being investigated to cost-effectively prepare for the future—pharmaceuticals, implant devices, cell and gene therapies, or some combination thereof. Gene therapy is one of the more promising possibilities because it seeks to correct the root of the problem, thereby minimizing treatment duration and cost. Currently, viral vectors have shown the highest efficacies, but immunological concerns remain. Nonviral methods show reduced immune responses but are regarded as less efficient. The nonviral paradigms consist of mechanical and chemical approaches. While organic-based materials have been used more frequently in particle-based methods, inorganic materials capable of delivery have distinct advantages, especially advantageous in orthopedic applications. The inorganic gene therapy field is highly interdisciplinary in nature, and requires assimilation of knowledge across the broad fields of cell biology, biochemistry, molecular genetics, materials science, and clinical medicine. This review provides an overview of the role each area plays in orthopedic gene therapy as well as possible future directions for the field.

Introduction

The imminent growth in the aging population will necessitate innovative therapies in orthopedic medicine. There are several different approaches to managing the wide variety of clinical pathologies. Tissue engineering therapies, specifically those incorporating gene therapies, have received increased attention, of late.^1–5 Some of the orthopedic pathologies targeted by gene delivery are nonunion fractures, complex fracture healing, and bone diseases such as osteoporosis and osteosarcoma.^6,7 The common thread in all of these ailments is inadequate bone regeneration, leading to significant losses in normal functions with concomitant pain.

Bone has a limited ability to regenerate and cannot heal when the defect size is above a certain threshold.^8,9 While grafts are currently considered the gold standard in treatments for large or complex fractures, there are problems associated with their use. Identifying proper sources for autografts is difficult because there are few anatomical sites where significant amounts of bone can be removed without the risk of donor-site morbidity.⁹ Allografts can be used, but immunosuppressive drugs are often required to prevent tissue rejection, which lends the recipient more susceptible to disease transmission.^9–11 Additionally, grafts from any source struggle to develop proper vascularization and therefore may fail to thrive.^9,10 Stem cell therapies under investigation require longer healing times and can be expensive. Several protein-based therapies, such as bone morphogenetic proteins (BMPs) have also been investigated, but they often fail to maintain adequate protein levels for healing.^12,13 BMP therapies, while highly touted, have been shown to be impractical for a number of reasons—(i) they are costly, (ii) extremely high dosages are required to be effective due to a short half life, (iii) dosage amounts have been determined using animal models and may not be suitable for human applications, and (iv) undesired ectopic bone growth may occur.^14–17 While BMP production is still the main target for orthopedic gene therapy, many of the aforementioned problems are overcome by using the cells to produce the BMPs in the gene therapy model.¹⁸

Previous review articles address the growing need for effective gene delivery strategies in orthopedics.^8,11,19 These reviews focus on pertinent growth factors, predominantly delivered using viral vectors.¹⁹ While the efficacy of nonviral vectors, in general, has been shown, the majority of reviews discussing their use only focus on organic delivery systems.^20,21 Inorganic materials have shown comparable and, in some instances, better transfection efficiencies than organic systems.²² However, there is no review that specifically discusses the role that inorganic nano-based materials will play in future orthopedic therapies. Inorganic materials have been shown to be resistant to microbes, are often associated with lower manufacturing costs, and have a longer shelf-life than organics.²³ Inorganic materials also offer unique electrical, mechanical, optical, and magnetic properties that are less common in polymeric particles.^24,25 The inorganic gene therapy field draws from various disciplines of science—cell biology, materials science, nanotechnology, and clinical medicine—to successfully design a complete vector system. This article is organized in the following sections: (i) a description of relevant growth factors in orthopedic applications; (ii) an overview of current gene delivery approaches; (iii) an outline of the biological mechanisms for inorganic gene delivery; and (iv) a discussion of the pros and cons of the potential inorganic systems and the modifications thereof. This review will complement the previous reviews by directly discussing the current and potential application of various inorganic nanoparticles in the orthopedic gene therapy field.

Growth Factors Involved in Orthopedic Therapies

It is important to have a fundamental understanding of the natural biological processes that occur during healing to mimic these processes in vivo with gene therapy. There are four critical issues of importance for bone repair:

1. The process is initiated by bioactive molecules, including cytokines and growth factors, which recruit osteoprogenitors and stimulate their differentiation.^6,11

2. A scaffold or matrix is needed for cell attachment and growth.^23,26

3. Osteoprogenitors and osteoblasts must have the necessary resources to synthesize and mineralize the osteoid matrix.

4. The area that is experiencing repair must have a rich vascular supply.

Bone grafts and gene delivery systems can participate in any or all of these elements. They may provide the cells, signals, scaffolds, or a combination thereof. Massive bone defects will require more time than smaller defects. The optimum duration or intensity of signaling depends on the gene transfer technique and the desired clinical outcome. There are risks of toxicity with prolonged exposure, so care must be taken to ensure that the delivery method is safe throughout the entire therapy time. It is also important to bear in mind that during normal healing processes, not all genes involved are activated at all times. There are times when some growth factors are being produced (the “on” state) and times when their production is suppressed, “off.” This “on and off” expression of genes should be mimicked during the therapy.⁹ For example, during normal healing of a fracture, different BMPs or angiogenic growth factors are upregulated at different stages of healing to direct physiologic healing.¹¹ It may be important in gene therapies to ensure that upregulation of specific BMPs is not prolonged and therefore encouraging heterotopic bone growth.²⁷

The most important cytokines used in orthopedic therapies belong to the class of BMPs that are able to induce differentiation in osteoprogenitors and have been shown to be key to bone formation.^8,9,12,28 The BMP family also plays a significant role in mesenchymal stem cell proliferation, chemotaxis, differentiation, and angiogenesis. BMPs direct and regulate the biological repair that occurs after trauma or disease; therefore, BMP production is an end-target for orthopedic gene therapies. Most current research involves direct BMP protein administration. While shown to be effective, they must be used at high concentrations as mentioned previously.¹³ BMP therapies may not yet be spatially and temporally identical to what happens in the body, but they have been shown to be effective in healing defects. There has been extensive research to support the success of BMPs in bone therapies^{6,8,11,12,29–31} such as promoting bone healing and endochondral ossification.¹³

There has also been evidence that other growth factors play a vital role in bone healing. Platelet-derived growth factor (PDGF) is locally released by platelets, macrophages, monocytes, and endothelial cells to increase DNA synthesis and cell proliferation. Vascular endothelial growth factors (VEGF) have been shown to promote angiogenesis. Insulin-like growth factor promotes cell proliferation and collagen type 1 production, an important matrix component. The family of transforming growth factor β, of which BMPs belong to, increases cell proliferation, angiogenesis, protein production, and specific matrix elements; therefore it contributes to the overall biomechanical strength of the resulting matrix. Kofron and Laurencin comprehensively reviewed a listing of growth factors and their influential effects in bone formation.⁸ Gersbach et al. provides a listing of possible combinations of growth factors for effective orthopedic regeneration.⁹ Some of the combinations that they describe are BMP and VEGF to enhance vascular growth, BMP-2 and Runx2 to enhance cell sensitivity, VEGF and receptor activator nuclear factor κB ligand (RANKL) to stimulate graft revitalization, among several others.

Growth factors that may aid osteoporotic patients have also been identified and are being investigated. The most notable growth factor is osteoclastogenesis inhibitory factor (OCIF, also referred to as osteoprotegerin).⁷⁰ As the name suggests, OCIF inhibits osteoclastic differentiation.⁷¹ Table 1 contains a summary of pertinent growth factors and their potential therapeutic applications.

Table 1.

Growth Factors for Orthopedic Therapies

Growth factor	Function	Therapeutic application	References
Growth factors for orthopedics
BMP-2	Induces cartilage and bone formation; induces osteoblast differentiation	Fracture healing, segmental bone defects, spinal fusion	^32–36
BMP-4	Directs early fracture healing; aids in callus formation	Fracture healing	^37–44
BMP-6	Induces osteoblast differentiation	Fracture healing	^33,45
BMP-7	Induces bone formation, induces osteoblasts differentiation	Fracture healing, segmental bone defects, spinal fusion	^35,36,46,47
BMP-9	Stimulates alkaline phosphatase activity in osteoprogenitors	Fracture healing	^48–50
FGF	Plays a role in endochondral development and intramembranous membrane formation; directs osteoblasts and precursors	Fracture healing	^51–54
IGF-I, -II	Promotes cell proliferation and collagen type I	Fracture healing	^29,55
PDGF	Increases DNA synthesis and cellular proliferation; upregulated in initial fracture sites; stimulates osteoblasts to produce IL-6, which recruits osteoclasts	Fracture healing	^3,29,56
TGF-β1	Increases cellular proliferation, angiogenesis, differentiation and protein production; promotes orthotopic bone formation by regulating mesenchymal cells, chondrocytes, osteoblasts and osteoclasts	Fracture healing	^29,43,57,58
VEGF	Stimulates blood vessel formation	Enhancement of fracture healing	^{29,39,59–65}
Transcription factors
LIM mineralization protein-1 (LMP-1)	Induces secretion of osteogenic factors, upregulates BMP expression and	Fracture healing	^8,66–69
OPG	Inhibits osteoclastic differentiation	Osteoporosis	^70–73
RANKL	Regulates osteoclastic resorption	Fracture healing, osteoporosis	^62,74,75
Runx2	Promotes osteogenic differentiation, downstream target of BMPs	Fracture healing	^76–80

BMP, bone morphogenetic protein; PDGF, platelet-derived growth factor; VEGF, vascular endothelial growth factor; IGF, insulin-like growth factor; RANKL, receptor activator nuclear factor κB ligand; OPG, osteoprotegerin.

Even though many of the growth factors critical to bone healing and osteoporosis have been identified, there are still problems associated with the high concentrations that must be used if they are to be directly administered as proteins.⁸¹ Administration of nucleic acids encoding for production of these growth factors allows for lower dosages to be introduced to the patient and therefore reduces possible toxicity or immunological complications.

Current Gene Delivery Approaches

Currently, viral vectors have been shown to be extremely effective in delivering therapeutic genetic segments to cells, but biological safety concerns remain. Their efficiency is viewed as the benchmark for a comparable gene delivery technique. To improve the efficiency of nonviral vectors, specifically those of the inorganic type, the basic cellular pathways these particles take must be better understood.

In general, there are two different strategies to effectively deliver nucleic acids (DNA and RNA sequences) to the proper cells or tissues—with a vector or without a vector.²⁰ Table 2 contains the main delivery methods currently available for gene therapy. Physical methods (including mechanical and electrical methods) are often used in conjunction with nanoparticles to transfect cells with plasmid DNA (pDNA).^20,82 These methods allow particles that normally could not traverse the cell membrane to enter into the cell.⁸³ The main limitations to these methods are that they are difficult to control and optimize in vivo.⁸⁴ Delivery methods that can infect larger, targeted populations are more practical for many gene delivery applications.

Table 2.

Methods for Gene Delivery

Method	Advantages	Disadvantages	Reference
Physical methods
Mechanical
Microinjection	Specific cells can be targeted	Only single cells can be targeted at a time; slow	²⁰
High Pressure	Particles or molecules normally unable to traverse the cell membrane can be inserted	Specific cells cannot be targeted	^84,105
Particle Bombardment	Particles or molecules normally unable to traverse the cell membrane can be inserted	Difficult to control the path of the particle; limited to superficial cells in vivo	^20,106,107
Magnetofection	Improved efficiency	Difficult to apply in vivo	¹⁰⁸
Electrical
Electroporation	Simple; efficient	Tissue damage; difficult to apply in vivo	^83,109,110
Vector-based methods
Viral
Adenovirus	Not dependent on cell replication	Brief infection	¹¹
Lentivirus	High efficiency	Modified from lethal viruses	¹¹
Retrovirus	Not dependent on cell replication	Possible insertional mutagenesis	¹¹
Nonviral
Organic	Degradable; easy synthesis	Low efficiency; byproducts may be toxic	²⁰
Inorganic	Differing degradability; easy synthesis	Low efficiency; non degradable materials may be toxic or accumulate in vivo	²²

Vectors, on the other hand, represent biological or chemical means to transport DNA. They fall into one of two categories—viral and nonviral. There are several significant differences between viral and nonviral methods. While viral methodologies are extremely efficient, they also inherently present a chance for viral infection.^85–89 Immunosuppressant agents are often required in conjunction with viral vectors to allow them to infect the host cell. Despite a virus being rendered replication deficient, it may still express viral proteins that may elicit an immune response and prevent efficient and lasting gene deliver. There have been several clinical trials involving viral gene delivery that have resulted in patient deaths or subsequent cancer.^85,86 Nonviral methods avoid these complications, but are less efficient in delivering DNA.

The nonviral vectors can be broken into two further subdivisions—organic and inorganic. As mentioned before, some of the organic vectors used to carry genetic segments are liquid buffers, proteoliposome carriers, synthetic polymer encapsulation, and hydrogels.^90,91 The organics used as gene delivery devices typically carry a cationic charge and are therefore able to condense the anionic DNA through electrostatic attraction.⁹² The surface groups of various polymers can be modified with specific signaling factors that allow for targeted cellular delivery via receptor-mediated endocytosis.⁹³ Both synthetic and naturally occurring polymers have been shown to be capable of transfection.⁹³ The degradation products of the natural polymers, such as chitosan, collagen, and gelatin, are less cytotoxic.⁹⁴ While this fact makes these natural polymers preferable, their efficiency is still less than most of the synthetic polymers.⁹⁵ Polymers can also be used in coordination with a tissue engineering scaffold, thus capable of also spatially regulating regeneration.⁹³ However, due to the lack of mechanical strength offered by these scaffolds, they would be unsuitable for applications where strength is required (i.e., load bearing applications).⁹⁶ In certain applications, the fast degradation times associated with organics is beneficial; in applications where it is necessary for the scaffold to maintain its integrity for prolonged times, they are not suitable. While significant improvements have been made in the organic field, their low efficiency rates and structural limitations (i.e., low strength and fast degradation) have restricted their application in orthopedics.

The inorganic materials that have been shown to be efficient DNA carriers are calcium phosphates (CaPs), gold nanoparticles (GNPs), silica, magnesium phosphates, and iron oxides.^97,98 The benefit of inorganic vectors mainly lies in their improved storage stability, cost effectiveness with respect to manufacturing, low immunogenicity, and their resistance to microbial attack.^22,97,99 Nanosized materials (<100 nm) have been shown to have an increased and beneficial biological response (e.g., enhanced osteoblast adhesion and proliferation and subsequent matrix formation) and have been shown to efficiently trap the DNA, subsequently allowing the DNA to escape the endosomes intact.^100–102 In regards to in vitro and ex vivo gene transfer, inorganics are superior to organics for attached cell lines owing to their increased density.^101,103 Due to gravitational forces, the inorganics settle onto the base of the tissue culture flask, the region containing the cells or tissues, thereby greatly enhancing their gene transfer efficiency by increasing adherence to the cell membrane.^101,104

There are a multitude of techniques and vectors that have been shown to have some degree of effectiveness in gene delivery. While viral vectors are currently more efficient than other methods, there have been many recent strides in the nonviral field.¹¹¹ The research and resulting modifications ofthe inorganics have shown promising results.¹¹² Inorganics have been shown to match growth factor production seen in organic nonviral vectors such as Polyfect^®, Superfect^®, and Lipofectamine™ (organic, standard lab agents).^113–116 They are thus able to carry the necessary transcription factors to produce a variety of growth factors.

As of yet, it is unclear as to what nucleotide should be used for each application. Previously, only DNA was utilized, but recent advances have shown messenger RNA (mRNA) to also be effective.¹¹⁷ While the two nucleotides may transcribe the same growth factor, they differ in several ways and thus one may be advantageous over the other. A brief discussion of the genetic transcription sequence will be discussed below, as well as the role that this process has in selecting which molecule to use.

RNA or DNA?

The general flow of genetic information can be summarized briefly as follows. A genetic sequence is activated on the DNA strand. The DNA is then transcribed into a corresponding mRNA strand inside of the nucleus. This strand of mRNA then exits the nucleus and enters the cytoplasm for translation into a protein in the ribosome. Ultimately, the biological goal for most orthopedic gene therapy applications is to produce the therapeutic protein—typically one or several from the BMP family—by specifying the genetic sequence that will activate its production. RNA-based therapies have also received increased attention.^117–119 mRNA has been shown to be able to induce transgene expression.^117,120 Alternatively, small strands of silencing RNA (siRNA) have been shown to suppress protein production contributing to a diseased state.¹²¹ Its potential has been demonstrated in vitro for diseases such as osteoporosis and osteosarcoma.¹²² There are certain advantages that RNA has over DNA, but there are several obstacles that must still be overcome if it is to be used clinically.^117,118,121

mRNA and siRNA operate in the cytoplasm, whereas DNA can function in the nucleus in the host genome.¹²³ mRNA will not integrate into the host genome, thus improving its biological safety by avoiding the genotoxic complications often seen with DNA usage.¹¹⁷ This means that the effect of the mRNA is transient versus the more permanent effect that DNA transfection has.¹²⁴ Depending on the application, this may be a beneficial difference. In applications where the therapeutic healing is only required for short durations (i.e., fracture healing), mRNA may prove to be more ideal than DNA segments. From a pure efficiency perspective, it has also been shown that mRNA transfection from electroporation into dendritic cells is about 1%–10% effective with DNA versus up to 95% with mRNA.¹¹⁷ This may have been due, largely in part, to the fact that the mRNA has less cellular obstacles to overcome than a DNA molecule does. Because the mRNA must only go to the ribosome for translation, the efficiency is most likely increased in this application. DNA, on the other hand, must be delivered to the cell nucleus and permitted through the nuclear pores to enter the nucleus. This fundamental difference of the cellular location where the therapeutic segments will operate is a significant difference between the two therapies.¹²⁴

In the past few years, siRNA has been researched extensively for genetic diseases and cancer.^121,125,126 siRNA operates in a different manner than mRNA does and thus has a few additional considerations. In short, siRNA molecules bind to the mRNA molecules in the cytoplasm that fuel the diseased state and render them unable to translate the protein causing the diseased state.^127–129 Just like mRNA and DNA segments, the siRNA must be transported into the cell with the aid of a vector. Several different materials such as polymers, liposomes, and CaPs have been shown to be effective vectors for siRNA delivery.^125,130

The main disadvantage with current RNA therapies revolves around the fact that RNA is much more susceptible to degradation.^131,132 If mRNA is to be used for gene therapy, the molecule must be protected from the enzymes present in the cytoplasm until it reaches the ribosomes. Additionally, mRNA, like DNA, has a net negative charge and thus cannot penetrate the cell membrane without the use of a carrier molecule. Several vectors that have been shown effective so far are cationic lipids and cationic polymers—DEAE-dextran, poly(L-lysine), dendrimers, polyethylenimine (PEI), and N-[1-(2,3-Dioleoyioxy)propyl]-N,N,N-trimethylammonium methyl-sulfate.^{131,133–137} Most of the considerations that are important for stable complexation between the vector and DNA are also necessary for RNA usage. These include, but are not limited to hydophobicity/hydrophilicity, molecular weight, charge density, pH of the solution during condensation, and electrostatic binding.¹³⁸ The extent to which nucleotide sequences bind to inorganic materials is predominantly driven by their surface area and surface charge. The surface charge is measured by the zeta potential of the material and is also pH dependent.

To optimize delivery, it is important to have an understanding of the pathway that nonviral vectors take to deliver the genetic information to the nucleus. The effectiveness of the vector at protecting and delivering the DNA/RNA at each stage, from introduction of the vector to transcription, ultimately affects the ability of the vector system to elicit the proper therapeutic effect.

Cellular Pathway for Inorganic Delivery

Nonviral delivery is similar to many drug delivery techniques. Complexes containing lipids, proteins, peptide, or polymeric carriers and ligands that target specific cell surface receptors can be used.¹³⁹ The endocytotic uptake can be enhanced by complexing the nucleotide with a cationic molecule that will be attracted to the negative charges on the cell membrane. Nucleotides are well-known to be anionic and therefore are often condensed with a cationic carrier. The complex, on the whole, is then positively charged. Alternatively, a ligand complex may also be used to target specific cells. The positively charged complex or ligand complex then binds to the negatively charged cell membrane and is endocytosed.¹⁴⁰

After the molecule has been taken into the cell, an endosome encapsulates the complex. Solubilization is achieved by lowering the endosomal pH.¹⁴¹ Due to the influx of protons, a charge gradient is formed between the endosome and the neighboring cytosol. This causes water to enter the endosome, resulting in its swelling and ultimate rupture. This causes an ejection of the nucleotide-vector complex. It is vital that the nonviral particle protects the DNA/RNA while in the endosome and allows its escape at the relevant time. The entire vector should be released before the complex begins to degrade.^23,140 Successful release from the endo-lysosomes does not necessitate high transfection efficiency because the genetic segment must still reach the nucleus if it is DNA.

The pathways that DNA takes once it has been released into the cytosol are vital to competent transfection. It is thought that the DNA travels toward the nucleus by way of diffusion and through the use of the cytoskeleton pathways. Diffusion is slow, however, due to the high viscosity of the cytosol. Molecular motors are used to actively transport endocytotic vesicles, such as endosomes and lysosomes, toward the nucleus.¹⁴² They are thought to do the same with genetic segments once they have been released from the endosome. Myosin is a type of protein filament that serves as the motor for directing motion along an actin filament.¹⁴² Dynein is a type of actin microfilament that is associated with providing the necessary intracellular movement of molecules inward (toward the nucleus).¹⁴² Because the actin-myosin scheme is an active transport system, it requires the use of an accessory molecule, dynactin.^21,142 It is beneficial for the endosome to be as proximal as possible to the nucleus before its degradation. If the endosome is degraded prematurely, the genetic segment may not be successfully transported to the nucleus. Lysomotropic agents, such as chloroquine, have been shown to be effective in increasing overall transfection efficiency because they prevent the lysosome/endosome from rupturing.²¹

Upon reaching the nucleus, the DNA can enter into the nucleus after crossing the nuclear pore complexes (NPC), which span the double-layered nuclear envelope.¹⁴³ This can be enhanced with nuclear localization signals.^140,144,145 The complex must effectively cross the NPC. Molecules can cross the membrane through the pores by three mechanisms: passive diffusion of species <10 kDa, active transport for molecules greater than 70 kDa and calcium regulated transport for molecules 10–70 kDa.^140,146 There are a variety of signaling agents that permit active transport of larger molecules.¹⁴⁷ Alternatively, calcium-regulated transport is of significant interest because it can make use of the elemental makeup of a vector used to facilitate entrance into the nucleus. An adequate calcium concentration in the cytosol keeps the calcium stores in the cisternae and blocks the release of inositol triphosphate (IP3), which is known to cause the NPC to close.^142,146,148 Thus, the presence of added intracellular calcium can help keep the NPC open. The general process by which a nonviral vector delivers the genetic segment is shown in Figure 1. (For more details on the mechanisms discussed, please see Refs.^20,149,150).

FIG. 1.

Cellular pathway for nonviral vectors.

If a delivery system using DNA as the genetic molecule is used, the DNA must be transcribed to mRNA in the nucleus. In order for this to happen, the DNA must be uncoupled from the vector, if it has not already done so; this may be accomplished through enzymatic activity once inside the nucleus.^151,152 The mRNA produced is dictated by which gene has become activated. The therapeutic activity is a result of the specific protein, growth factor, hormone, etc. that is coded for in the mRNA. The genes initiate a cascade of events by producing proteins and releasing cytokines. As mentioned previously, some of the growth factors used for orthopedic applications include the family of BMPs, PDGF, FGF, and VEGF as well as the transcription factors Runx2 and RANKL.²⁹ (See Ref.¹⁵³ for a complete listing of growth factors delivered in experimental models for bone healing). Some of these genes may encode only one growth factor or the production of one factor may trigger the synthesis of multiple growth factors.¹⁵⁴ It is clear, however, that combinations of these different growth factors will be the most effective treatment because their synergistic effect most closely emulates the natural repair mechanisms of the body.^9–12,155

Potential Inorganic Vectors, Their Pros and Cons, and Modifications

Based on the cellular pathway that a vector must take, there are several stages that would benefit from further vector modification. The goals of modification are to increase transfection. This can be accomplished by promoting cellular uptake via ligand–receptor complexes, increasing the positive charge of the complexes, or enhancing stabilization to protect the DNA from degradation in the endosomes and lysosomes. There are also opportunities to increase the efficiency through our understanding of entrance mechanisms to the NPC. For each of the materials listed below, and as summarized in Table 3, there will also be a brief discussion of successful modifications to the vector system.

Table 3.

Summary of Inorganic Carriers for Gene Delivery

Material	Advantages	Disadvantages	References
Iron oxides	Capable of magnetofection	Difficult to translate in vivo	^{103,114,156–165}
Silica	Biocompatible, easy to surface functionalize, mesoporous	No known mechanism to traverse the NPC, ultimate particle fate is unknown	^{102,114,166–169}
Gold	Easy to surface functionalize; photothermal properties; may be used in combination with gene gun techniques	Nondegradable, may accumulate in the liver	^170–178
QD	Fluorescent, small size	Nondegradable, may accumulate in the liver, some toxicity	^179–182
Mg- PO₄	Biodegradable, biocompatible	Limited research has proven their efficacy for multiple cell types	¹¹⁵
Ca– PO₄	Biodegradable, biocompatible, lanthanides can be incorporated to yield fluorescence	Difficult to control synthesis	^{116,140,169,183–193}

NPC, nuclear pore complexes; QD, quantum dot.

There are two fundamental categories of the inorganic vectors-biodegradable and nonbiodegradable. Many organic materials are also biodegradable, but their degradation products are toxic. Apatites, such as calcium and magnesium phosphate materials, are biodegradable and their byproducts are biocompatible. Iron-based nanoparticles, GNPs and silica nanoparticles do not degrade and care should be taken that they will not accumulate to toxic levels during in vivo therapies. This difference is critical in ultimately choosing a vector system based on the amount and duration of gene therapy required.

Iron-based nanoparticles

Magnetic nanoparticles have shown efficacy in transfecting a variety of cell lines^156–161 as well as being capable of delivering pDNA and siRNA.^{125,156,162,163} These nanoparticles are relatively simple to produce and can be complexed or functionalized with either viral or nonviral vector systems. Application of a magnetic field either in vitro or in vivo allows spatial targeting and has shown improved transfection. Magnetically induced or aided transfection is referred to as magnetofection. These delivery systems do not rely on cellular machinery to traverse the cell membrane, therefore cells that are normally incapable of being transfected (i.e., nondividing cells) could be manipulated using this method.

Iron-based nanoparticles have shown good storage stability and can be prepared to tailor a variety of applications. Fe₃O₄ nanoparticles containing enhanced green fluorescent protein (EGFP) DNA have been shown to transfect mouse osteoblasts with the application of a magnetic field increasing efficiency.¹⁶⁴ Surface modified iron oxide particles have shown improved transfection due to minimizing coagulation of particles, providing a positive particle surface charge, and multiple binding sites for ligands.¹⁶⁵ While many of these surface modifications are not unique to iron-based nanoparticles, it is important to note that the modifications have functionally behaved as hypothesized for this material.

Silica

Silica particles have a negative surface charge and are, therefore, often modified with cationic surface molecules. They have been modified with DMRIE-C,^103,114 aminoslkylsilanes AEAPS and AHAPS,^102,166 and sodium chloride.¹⁶⁷ These surface modifications give the silica nanoparticle complexes the necessary cationic charge for cellular entry.

Organically modified silica nanoparticles are thought to improve efficiency through protecting the DNA from enzymatic degradation and have been shown to transfect osteoblasts at levels comparable to lipofectamine.^168,169 The main advantage of organosilicates is that they are nontoxic to cells in vitro where many organic-based materials have been shown to exhibit toxicity. Ca-doped organosilicates show improved selectivity in transfecting osteoblasts as compared to hepatocytes, suggesting that the addition of calcium enhances transfection to osteoblasts.¹⁶⁸

Gold nanoparticles

GNPs have been surface functionalized by adding mixed monolayer protected gold clusters,¹⁷⁰ and inserted by gene gun technology.^171–173 Aminealkyl thiols have also been added to GNPs and shown to be effective for pDNA delivery.¹⁷⁴ Transfection efficiency was shown to be related to the ratio of the modified nanoparticle to the pDNA, the positive charge density on the surface, and the length of the alkyl chain. Several groups have used PEI to enhance the cationic delivery of the GNPs and demonstrated improvement over PEI alone^85,175 GNPs exhibit a highly tunable surface and therefore can be used for siRNA and mRNA applications where stability is of utmost importance.¹⁷⁶

GNPs have photothermal properties that could be exploited for delivery applications. When they are irradiated with light 800–1200 nm in length, they are locally exothermic. They have been shown to be effective in selective destruction of cancer cells and in releasing macromolecules from a gold shell after laser excitation.^177,178 Thus far, there is no reported use of GNPs in orthopedics, but their potential use in the nonviral field has been shown.

Quantum dots

Quantum dots (QD) are inorganic nanoparticles that exhibit strong fluorescence signals and have shown promise as gene delivery vehicles.¹⁷⁹ Their fluorescent properties make them a unique carrier because they can be imaged in vivo without further modification. In comparison, organic dyes can also be conjugated to various nanoparticles and used for imaging purposes. They do not offer the full range of innate capabilities that QD possess and are generally considered to have increased toxicity.

Typically, QD are <50 nm and can easily be functionalized with various biological molecules such as DNA, siRNA, proteins, and peptides.¹⁸⁰ They have been shown to successfully transfect mammalian cells with EGFP.¹⁸¹ They have also been used in targeted silencing of SKBR3 breast cancer cells overexpressing human epidermal growth factor receptor 2 (HER2) by being conjugated with HER2/neu siRNA.¹⁸² Another study showed the efficacy of QD delivery of matrix metalloproteinase-9 siRNA to brain microvascular endothelial cells—the main component of the blood brain barrier.¹⁸⁰ Although QD have not yet been complexed with nucleotide sequences specifically related to orthopedic pathologies, their demonstrated ability to transfect several different cell types is encouraging.

Magnesium phosphates

Many inorganic nanoparticles can be used in gene delivery systems, but remain relatively unexplored. Magnesium phosphate nanoparticles have been shown to be efficient by forming a cationic complex with the DNA, thus allowing the molecule to cross the cell membrane through ion channel-mediated endocytosis. Magnesium behaves similarly to calcium because it is also a divalent ion. It is also a constituent in body fluids and therefore shows high biocompatibility. Magnesium phosphates were shown to efficiently transfect DNA as efficiently as Polyfect in vitro to HeLa cells.¹¹⁵ While there has been no work specifically showing the efficacy of magnesium phosphate nanoparticles for delivering orthopedic-related genes, this material has shown that it is capable of delivering therapeutic segments.

Calcium phosphates

Of all the inorganic vectors, CaP-based materials may be the most promising for orthopedic pathologies. CaPs have inherent material properties that make them advantageous owing to the materials' similarity to mammalian bone and the importance of calcium in many cellular processes. Calcium is a relatively strong cation and therefore attracts the anionic DNA, allowing for complexation. In addition, CaPs provide the necessary cationic surface charge to enter the cell via endocytosis. The surfaces of CaPs can be modified and have shown the ability to promote longer blood circulation and to target specific tissues. The size and shape of CaPs can be controlled during synthesis and tuned for the application.^140,183,184 CaPs have been shown to increase transfection efficacy as compared to pDNA transfection in vitro and in vivo.^185,186

On the cellular level, the CaP system has several innate material properties that likely aid in efficient transfection. One of the main obstacles encountered in the intracellular environment is the activity of nucleases in the cytoplasm. In the CaP system, the pDNA forms a rigid complex with the CaP and protects the DNA from these nucleases, which allows a greater amount of intact DNA to reach the nucleus.^{116,187–189}

One of the possible mechanisms by which the DNA is released from the CaP is through the slow release of protons into the endosomes.^141,190 The pH in the endo-lysosome is slowly lowered by ATP-dependent proton pumps. It has been shown that at higher pHs, the DNA remains attached to the CaP system, but that the pDNA is released from the vector at a lower pH.¹⁸⁷ It has also been suggested that the presence of protonable groups within the endosome allows for the vector to be released into the cell without degradation, generally referred to as the proton sponge theory.^21,142 Although it has not been explicitly suggested, it is thought that the phosphate present in the CaP system may help to buffer the endosomal pH and thus prevent DNA degradation from pH changes.

Lysomotropic agents are often used to prevent the degradation of the endosome so that the cytoskeleton is able to move the endo-lysosome closer to the nucleus before release. Rupture of the endo-lysosome arises from the influx of water ions resulting from the voltage gradient.¹⁴² The longer that the endo-lysosome can resist this rupture, the closer it will be to the nucleus. If the phosphate is buffering the solution, rupture will be prevented until the endo-lysosome is close to the nucleus. It is imperative that upon reaching the nucleus, there is a way for the pDNA to cross the nuclear envelope. As was discussed previously, intracellular calcium concentrations affect the permeability of the NPC. High intracellular calcium concentrations are associated with retaining calcium in the cisternae and blocking the release of IP3, which closes the NPC.¹⁴⁸ If CaP is used as the delivery mechanism, there exists a high concentration of calcium in the cell,¹⁹¹ thus allowing the pDNA (if it has been detached from the CaP) to enter the nucleus through the NPC. Thus, in the CaP system, the pDNA will be delivered to the doorstep of the nuclear envelope, with the NPC open; this is the optimal scenario for a nonviral vector.

There have been multiple studies that prove the efficacy of CaP as an inorganic vector. Roy et al. used a co-precipitation method to complex the pDNA with CaP nanoparticles.¹⁶⁹ The CaP system was employed both in vitro and in vivo successfully and the target genes were expressed in major organs of the mice models more so than the pDNA injected in its naked form. Welzel et al. successfully transfected CaP nanoparticles coupled with DNA into human endothelial cells.¹⁹² Bisht et al. demonstrated that the CaP nanoparticles showed better transfection efficiency than the commercial transfecting agent Polyfect.¹⁸⁸ CaPs have also been used to successfully silence GFP using siRNA molecules.¹³⁰ While none of these preliminary experiments demonstrate CaPs potential in orthopedic gene therapies, their ability to be used as vectors in a variety of cell lines has been demonstrated.

CaP nanoparticles with complexed DNA may also be incorporated into tissue engineering scaffolds.¹⁹³ After the DNA has been released, the degraded particles may be used as ion sources for new bone growth. This is a property that it is obviously unique to CaPs and makes them ideal candidates for orthopedic pathologies.

Concluding Remarks

While there exists a variety of inorganics for gene delivery applications, there has been no review that compares the various inorganics for orthopedic applications. As was demonstrated in this review, the class of CaPs seems to have the most inherently beneficial biological aspects—namely, the presence of calcium in the vector. Calcium may serve to improve selective transfection to osteoblasts, provide a positive charge for improved adhesion to the cell membrane and therefore cellular entry via endocytotic uptake, and maintain intracellular calcium levels so that the NPC remains open. CaP nanoparticles complexed with nucleotides may easily be integrated into bone tissue engineering scaffolds to provide both the scaffold and signaling agents necessary for efficient repair and regeneration.

Inorganics, as a class of materials for gene delivery, have many benefits over organic vectors—their resistance to microbial attack, storage stability, reduced cytotoxicity, and unique material properties. The field remains young but these diverse materials will undoubtedly find their niche in the gene therapy field. This review has provided the groundwork, both from a biological perspective, as well as a materials perspective on areas where the efficiency may be increased. By deepening our understanding of the cellular pathway that these particles take, appropriate modifications to the material or delivery method may be made to enhance their efficacy. Inorganic materials show much promise as efficient vectors for orthopedic gene delivery.

Footnotes

Acknowledgments

The authors wish to thank Joseph Lawrence for his critical reading of this article. This work was funded by NSF CMMI 0753479.

Disclosure Statement

No competing financial interests exist.

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