Background

As discussed earlier, the direct injection of naked, unmodified siRNA was not found to be highly effective as siRNA will suffer from many challenges such as RNA degradation, very short half-life and circulation time, weak targeting and biodistribution, etc. Therefore, in order to enhance its therapeutic efficacy and make full use of its capabilities and unique characteristics, siRNAs should be encapsulated in special delivery carriers—the nanoparticles.

It is necessary to note before discussing siRNA-loaded nanoparticles the fact that many scientists have tried modifying the siRNA before injecting it in hopes of enhancing its therapeutic effect and overcoming the barriers that stop it from reaching clinical applications. Chemical modifications performed on siRNA backbone, especially at the 2′ Ribose sugar, allow activity enhancement and prolonged half-life without function alteration. Moreover, modifications done at some linkages such as phosphorothioate and boranophosphate can lead to increased efficiency of siRNA [40]. However, despite all efforts in modifying siRNA to overcome all possible barriers, it remains poor and unable to succeed in clinical trials due to the fact that excessive modification can alter the molecule’s function and biodistribution.

There are countless modifications that can be performed to the siRNA molecule, touching the backbone, sugar (ribose), phosphates, bases, nucleotides, etc. For instance, substitutions in the ribose sugar can help the siRNA molecule overcoming degradation. However, replacing about 50% of nucleotides, especially in the 2′ position, can severely interfere with the silencing process by increasing the molecule’s thermal stability. As a consequence, this can prevent the dsRNA that were broken down into simpler and smaller fractions from forming complexes with RISCs. Therefore, the mRNA will not be divided and the silencing or downregulating action of siRNA will be inhibited [41]. Nanoparticles are emerging as fascinating tools not only for bio-imaging (diagnosis) as previously considered but also for therapy by acting as specific carriers to accurately and safely deliver siRNA to the appropriate target sites. However, not all existing nanoparticles are ideal candidates for this mission as they must possess the correct formulation, length, size, shape, charge, etc., and must be successfully able to: (1) protect and fortify the cargo—siRNA, (2) have low toxicity, (3) avoid triggering an immune response, (4) accurately deliver and release the cargo at the target site, and (5) preserve the cargo’s physiology and function [42].

Effects of Nanoparticle Modification on Delivery of siRNA

Since nanoparticles should meet specific rules or conditions to be able to successfully deliver siRNA to the target site—which can seem impossible to satisfy—their formulation has to be modified to suit these conditions and boost the success rate of the treatment. The nanoparticles’ modifications will be briefly discussed.

Size and Shape: Size plays an important role in the nanoparticle’s pharmacokinetic behavior where medium-sized particles about 50 nm in diameter were reported to have the highest levels of cellular uptake compared to nanoparticles having a 14 or 75 nm diameter [43].

In addition, nanoparticles’ shape greatly affects the cellular internalization. In a study comparing round to rod-shaped nanoparticles, round nanoparticles were found to have five times more cellular internalization compared to their rod-shaped counterparts. This is due to the fact that the time and effort required for the cells to completely wrap up the rod-shaped nanoparticles by their membranes and absorb them was far more than the round nanoparticles [44]. Relating what has been reported to lung cancer therapy, most nanoparticles are deposited in the lungs through mechanisms like sedimentation and diffusion due to the lung’s physiology. As a consequence, nanoparticles having a larger aerodynamic diameter (D_a)  5 μm are deposited in the upper airways (far away from tumors in deeper segments and lobes) compared to those having a smaller diameter 1 μm  D_a  4 μm that are deposited at deeper parts of the airways. However, this does not mean that the smaller the size the better since particles with a diameter 1 μm get exhaled outside the lungs [45–48].

Charge: Surface charge is another important factor that affects nanoparticle’s pharmacokinetic properties. The use of either negatively or positively charged nanoparticles is heavily case-dependent. From one side, cell membranes are usually negatively charged, thus negatively charged nanoparticles tend to repel and will be unable to move inside the cells or even cross their membranes. In this case, positively charged nanoparticles will be preferred to deliver the siRNA [49]. Studies involving Poly Lactic acid—Poly Ethylene Glycol (PLA-PEG) nanoparticles covered with stearylamine—a cationic (positively charged) lipid—showed increased internalization in HeLa cells compared to PLA-PEG nanoparticles without stearylamine coating (i.e., without a positive charge) [50]. From the other side, cationic nanoparticles might cause problems such as reducing membrane integrity, damaging mitochondria and lysosomes, etc. Therefore, anionic nanoparticles will be preferred in these conditions. Moreover, phagocytic cells prefer interacting with anionic nanoparticles more than their positively charged nanoparticles [51].

It is also crucial to note that nanoparticle size, charge, and shape are not the only conditions one should pay attention to. Experimental conditions such as the buffer used, the medium in which the experiment was conducted, the temperature, and the pH are also important factors [52]. In most cases, even a simple variable can be game-changing: Carboxy (PS-COOH) and amino-functionalized polystyrene (PS-NH₂) having the same size (100 nm) but conducted in different experimental conditions showed different internalization methods [53]. In addition to that, the interaction between nanoparticles and cell membrane not only depends on the nanoparticle size, but also on the membrane wrapping process that starts off endocytosis. Therefore, nanoparticles having a small size and less receptor—ligand interactions need to be close enough to the cell to initiate membrane wrapping and vice versa [54].

Hydrophobicity is another important factor that should not be ignored. For some cells, hydrophobic nanoparticles can be stuck in the bi-layer, whereas semi-hydrophilic nanoparticles can be absorbed into the membrane [55]. In another study, dichain nanoparticles modified by DMAB showed greater interaction and cellular internalization than their single chain versions. This is due to the fact that having two hydrophobic chains lead to more interaction than single chains CTAB (Cetyl-Trimethyl-Ammonium Bromide) and DTAB (Dodecyl-Trimethyl-Ammonium Bromide) [56]. Therefore, the choice of hydrophobic, hydrophilic, and/or semi-hydrophobic nanoparticles heavily depends on the cell type.

Types of Nanoparticles Used for siRNA Delivery to the Lungs:

Organic Nanoparticles

Lipid-Based Nanocarriers

They include all cationic (positively charged) lipid nanoparticles, lipid-like substances, and liposomes. What makes lipid-based nanoparticles good candidates for siRNA delivery is the fact that they are effective carriers that can be easily modified and functionalized. Moreover, lipids are known for their good interaction with the negatively charged cell membranes. However, one major drawback of these nanoparticles for prospective clinical applications is their biocompatibility but not their inherent toxicity: many researched siRNA loaded lipid-based nanoparticles used for the treatment of lung cancer have not been approved and failed to be commercially available due to their toxicity caused by the indirect activation of cytokine. Two of the aforementioned compounds are Oligofectamine and Lipofectamine [57].

Polymer-Based Nanocarriers

Polyethyleneimine (PEI) polymers are considered the best polymer-based nanoparticles for siRNA delivery thanks to their positive charge, molecular weight, and special branching pattern. PEIs have unmatched transfection efficiency but they are also limited in use due to their cellular toxicity in many cell types. Fortunately, this toxicity can be reduced by the addition of hydrophobic, hydrophilic groups, or both. Experiments involving PEIs modified with both hydrophobic and hydrophilic groups have shown reduced cytotoxicity in mouse models using intratracheal delivery method [58].

Dendrimers are also used for siRNA delivery due to their various nanoparticles—cargo binding mechanisms which include adsorption, chemical conjugation, and encapsulation. Polyamidoamine (PAMAM) dendrimers have been studied on a large scale for many years as a potential carrier for siRNA. PAMAM advantages include the easy binding to siRNA thanks to their surface amines and also the efficient escape of the cargo at the appropriate target site(s) with the help of their internal amines. A study on modified PAMAM showed less densely packed dendrimers when PAMAM had a triethanolamine core leading to increased internalization of siRNA [59].

Inorganic Nanocarriers

Inorganic nanocarriers have fascinated scientists due to their wide potentials. They are not only limited to metallic nanoparticles but also involve many sub-types such as semiconductor nanoparticles, Carbon-based nanoparticles, silica nanoparticles, quantum dots, fullerenes, etc. These nanoparticles have long astonished researchers and scientists due to their two-in-one role as both carriers for siRNA for lung cancer therapy and bio-imaging tools used in diagnosis and accurately tracking the siRNA trajectory upon delivery and its activation at target sites. Despite the debate regarding the toxicity of metal-based nanoparticles, new studies have shown that they can be modified like other nanoparticles easily to reduce their toxicity levels. Moreover, their benefits that include stability, noninvasive fluorescent nature, and controllability might outweigh their disadvantages and drawbacks [60].

Gold Nanocarriers:

Gold nanoparticles have been studied extensively throughout the years as the ultimate candidates for siRNA delivery. Their special surface plasmon resonance (SPR) characteristic makes them beneficial for bio-imaging, and in addition to their stability and efficient delivery of siRNA, a lot of effort is being put to exploit the true potential of these particles and further improve their biocompatibility for prospective clinical applications [61].

Iron Oxide Nanocarriers:

Many metal oxide particles, especially iron oxide particles, have taken the spotlight due to their unique characteristics. In addition to the fact that these nanoparticles can be used for bio-imaging, have excellent cellular absorption, are stable and modifiable like other metal-based nanoparticles, they have the distinguishing feature of thermal activation that is also shared with gold nanoparticles. Iron oxide particles can be used to heat the tumors to lethal temperatures causing the coagulative necrosis of tumor cells upon the application of alternating magnetic fields [62]. Furthermore, SPIO nanoparticles were reported to be perfect candidates for future siRNA therapies for many diseases including lung cancer given their strong contrast (i.e., MRI signal) and their unique magnetic properties which allow them to be guided using an external magnetic field to accumulate in the tumor sites [63]. Magnetic targeting can improve both the delivery of siRNA and/or therapeutic compounds (i.e., chemotherapeutic drugs) to improve cancer treatment [64]. We have previously reported that targeting of intravenously injected SPIO nanoparticles to the lung was proved to be enhanced when using external high-energy magnets positioned over a specific region of the lung [65]. This approach was further elaborated in another study in which the use of high-energy magnets offered improved theranostic effect of Doxorubicin-loaded iron-tagged nanocarriers, by magnetically targeting them towards metastatic tumor sites in the lungs [66].

Delivery Mechanisms of siRNA Loaded Nanoparticles for Lung Cancer Treatment

Intratracheal Delivery

It is one of the most widely used methods for drug delivery to the lungs. In general, most studies on siRNA-loaded nanoparticles carried on animal models use intratracheal siRNA delivery techniques, however, it is not used in humans. Although this technique provides negligible loss of therapeutic material, high efficiency, promptness, and low cost, but its major drawback is the surgical procedure that has considerable risk and is uncomfortable for the patient [67]. As an example, PEG-coated nanoparticles modified by Arginine-Glycine-Aspartic acid (RGD) peptide loaded with mouse c-myc siRNA administered through intratracheal route showed successful downregulation of c-myc gene expression and stopped tumor proliferation with little loss of therapeutic material [68].

Intranasal Delivery

What makes intranasal delivery superior to intratracheal delivery is that it can be used in humans. In fact, many commercially available drugs are being used in humans to treat diseases like asthma and respiratory infections and are available in the form of sprays and nasal droplets. Delivery of siRNA through these devices is painless, however, humans cannot be compared to the mice or rats used in pre-clinical studies. The reason behind that is the fact that animals breathe mostly through their nose compared to humans, in addition to that, the lung anatomy and pathway differ between humans and animals so the quantity of therapeutic material delivered to the target site through this method will be relatively low. An experiment conducted on normal adult volunteers showed that upon inhalation of mono-disperse particles via intranasal delivery, only 3% of the particles reached the lungs and the remaining 97% remained in the nose due to them being captured by the nasal hairs and cilia [69].

Intravenous Delivery

Although it possesses multiple problems and challenges, intravenous siRNA-loaded nanoparticles remain the most practical and most applicable method for delivery in humans. The problem is that upon administration of this compound, it will not reach the target organ or site directly and at the same amount as expected, instead, it undergoes multiple passes and circulations in the body and distributes unevenly in multiple organs leading to little accumulation in the target organ or site. Also, during its circulation, the compound undergoes filtration and elimination by the liver and excretion by the kidneys which shorten the circulation half-life and reduce the efficiency of the treatment. Fortunately, researchers have proposed a “trick” to overcome this problem. The secret lies in the use of sticky siRNA (ssiRNA): in vivo injection of ssiRNA and PEI successfully lead to the downregulation of tumor proteins, blockage, and prevention of tumor growth [70].

The strengths and pitfalls of each delivery method previously discussed are summarized in (Figure 4).

Figure 4. Scheme showing the advantages, disadvantages, and opportunities of some delivery methods for siRNA loaded nanoparticles to the lungs. Adapted from Reference [42].

Barriers of siRNA-Loaded Nanoparticles Delivery to the Lungs

Nanoparticles carrying siRNA must overcome multiple barriers both extracellular and intracellular to prove themselves worthy of becoming the next generation therapy tool for the treatment of multiple chronic and fatal diseases including lung cancer.

Extracellular barriers can be classified as biological, chemical, and physical barriers which are all related. One of the many barriers includes opsonization where foreign particles originating from outside the body called “antigens” are destroyed by the immune system by phagocytes and macrophages. Moreover, siRNA conjugated nanoparticles delivery to the lungs is more challenging compared to other organs due to the high number of immune cells (phagocytes and macrophages) that are proliferating in the lungs. It is worth noting that that the average human breathes more than 1,000,000 viruses and airborne bacteria, in addition to the toxic particles due to air pollution, therefore, the lungs are exposed to a wide variety of antigens and must be reinforced and protected more than any other organ by macrophages and phagocytes. One proposed method to overcome this issue involves either making the injected therapeutic compound somehow “invisible” to the immune cells by modifying the surface of the nanoparticles or decreasing the interaction between the nanoparticles holding the siRNA and other particles.

Intracellular barriers involve the inaccurate delivery, escape or leak, or inadequate packing of siRNA into the nanoparticles. Endocytosis poses a big problem that stands in the way of siRNA accurate delivery where the nanoparticles containing the siRNA can be phagocytosed and released at random sites different from the desired site. Moreover, the reticuloendothelial system (RES) can pose a threat to the accurate delivery of siRNA via the nanoparticles whereupon administration, these particles are directly transported to the RES organs such as spleen and liver to be later excreted by the kidneys in the body’s natural act of removing all unwanted antigens from the body that could be harmful. Therefore, the siRNA accumulates in the liver and spleen instead of the target organ and could escape the nanoparticle capsule upon phagocytosis due to inappropriate packing [71].