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Gene therapy using vectors has attracted attention in recent years for the treatment of cancers caused by gene mutations. Besides, new treatments are imperative for lung cancer, including non-small cell lung cancer (NSCLC) and malignant pleural mesothelioma (MPM), due to its high mortality. We developed a minimally invasive and orally inhalable tumor suppressor gene drug (SFD-p16 and SFD-p53) with non-viral vectors for lung cancer treatment by combining tumor suppressor genes with an inhalant powder that can deliver active ingredients directly to the lung. We used NSCLC (A549 and H1299) and MPM (H2052) cell lines in an air–liquid interface culture. Transfection of A549 and H2052 cells with SFD-p16 significantly increased p16 mRNA expression levels and decreased cell proliferation in both cell lines. Similar results were obtained with transfection of H1299 with the inhalable gene drug SFD-p53. In an in vivo experiment, a mouse model of lung cancer with orthotopically transplanted luciferase-expressing A549 cells was subjected to intratracheal insufflation of SFD-p16. Consequently, SFD-p16 effectively and directly affected lung cancer. This study suggests that inhalable gene drugs are effective treatments for NSCLC and MPM. We expect inhalable gene drugs to present a novel gene therapy agent for lung cancer that patients can self-administer.

Inhaled lung cancer therapy is promising because of direct and noninvasive drug delivery to the lungs with low potential for severe systemic toxicity. Thus chemotherapeutic drugs have been administered clinically by nebulization of solution or suspension formulations, which demonstrated their limited pulmonary absorption and relatively mild systemic toxicity. In all these clinical trials, however, there was no obviously superior anticancer efficacy in lung cancer patients even at the maximum doses of drugs limited by pulmonary toxicity. Therefore methods that deliver both higher anticancer efficacy and lower pulmonary toxicity are strongly desired. In addition to the worldwide availability of pressured metered dose inhalers (pMDIs) and dry powder inhalers (DPIs) to treat local respiratory diseases, recent innovations in medicines and technologies are encouraging next steps toward effective inhaled lung cancer therapy with new therapeutic or drug delivery concepts. These include the discovery of target cells/molecules and drug candidates for novel cancer therapy, the development of high-performance inhalation devices for effective pulmonary drug delivery, and the establishment of manufacturing technologies for functional nanoparticles/microparticles. This review highlights the present situation and future progress of inhaled drugs for lung cancer therapy, including an overview of available inhalation devices, pharmacokinetics, and outcomes in clinical trials so far and some novel formulation strategies based on drug delivery systems to achieve enhanced anticancer efficacy and attenuated pulmonary toxicity.

Gene therapy is a breakthrough treatment strategy against several intractable and lethal diseases that previously lacked established treatments. Viral and nonviral vectors have been studied to realize higher gene transfection efficiencies and to suppress the degradation of gene by nucleolytic enzymes in vivo. However, it is often the case that the addition of a vector results in adverse effects. In this study, we identified formulations of dry naked plasmid DNA (pDNA) powders with no vector showing significantly higher gene expression than pDNA solutions including vectors such as polyethylenimine (PEI) in the lungs of mice. We prepared the naked pDNA powders by spray-freeze-drying with various excipients. The gene expression of naked pDNA powders exceeded those of pDNA solutions containing PEI, naked pDNA solution, and reconstituted pDNA powder. Gene expression of each naked pDNA powder was dependent on the composition of excipients. Among them, the mice that were administered the pDNA powder composed of low-molecular-weight hyaluronic acid (LHA) as an excipient showed the highest gene expression. The lactate dehydrogenase activity and concentration of inflammatory cytokines in bronchoalveolar lavage fluid were comparable to those caused by ultrapure water. The results suggest that useful dry naked nucleic acid powders for inhalation could be created by optimizing the excipients, offering new insights into the development of pulmonary gene therapy.

In the development of a drug for intra-articular administration, a sustained-release formulation is desirable since it is difficult to sustain the effects of conventional injections due to fast drug leakage from the joint cavity. In this study, we prepared sustained release gel formulations for intra-articular administration containing indocyanine green (ICG) as a model drug to follow its fate after intra-articular administration in rats with in-vivo imaging system (IVIS). ICG administered as an aqueous solution leaked from the joint cavity in a short time and was excreted out of the body within a day. On the other hand, ICG in the sustained-release formulations was retained and released in the joint cavity for a week. Next, we prepared a sustained-release formulation with hyaluronic acid (HA) as the gel base containing a pain-relief drug (Drug A). We had administered it and other formulations into the rat knee where we injected bradykinin to evaluate their walking distance after 1 and 3 d. The effect of an aqueous solution of Drug A disappeared on day 3. The HA gel formulation without Drug A was more effective than the aqueous solution. The HA gel formulation with Drug A was the most effective; the walking distance was about 85% of the baseline on day 3. This study showed that the gel formulations were effective to sustain the release of a drug in the knee joint, and that the combination of a pain-relief drug with HA gel was effective to improve the mobility of the acute pain model rats.

We performed in vivo/ex vivo/polyacrylamide gel electrophoresis (PAGE) fluorescence imaging of near-infrared fluorescence (NIRF)-labeled siRNA (Cy5.5-siGL3) in mice to investigate the validity of each fluorescence imaging result as the biodistribution/biostability assessment of siRNA. Statistically significant correlations could be obtained between the in vivo and ex vivo fluorescence intensities of Cy5.5 in the relevant regions/tissues, except the lung region/tissue after intravenous administration. On PAGE fluorescence images with the naked formulation, there was no band corresponding to intact Cy5.5-siGL3 from all the tissues evaluated after intravenous administration, indicating that the fluorescence detected by in vivo and ex vivo fluorescence imaging was derived from degraded Cy5.5-siGL3 or free Cy5.5 cleaved from Cy5.5-siGL3. However, the band was detected from the lungs after intratracheal administration of the naked formulation, confirming higher stability of siRNA on the respiratory epithelium than in the blood. Regarding the polyethyleneimine formulation, the band was detected from all the tissues evaluated after intravenous administration and from the lungs after intratracheal administration, verifying the enhanced stability of siRNA in the body. These results clearly indicated the necessity of comprehensive analysis from in vivo/ex vivo/PAGE fluorescence imaging to precisely assess the distribution and stability of NIRF-labeled oligonucleotides including siRNA in the body.

In order to evaluate the in vivo effect of inhaled formulations, it is a gold standard to create a lung metastasis model by intravenously injecting cancer cells into an animal. Because the cancer grows from the blood vessel side, there is a possibility of underestimating the effect of an inhaled formulation administered to the lung epithelium side. In addition, the metastasis model has disadvantages in terms of preparation time and expense. The present study aimed to establish a new method to evaluate the effect of an inhaled small interfering RNA (siRNA) formulation that is more correct, more rapid, and less expensive. We investigated whether siRNA can suppress gene expression of plasmid DNA (pDNA) by serial pulmonary administration of siRNA and pDNA powders prepared by spray-freeze-drying. We revealed that formulations of dry siRNA powder significantly suppressed gene expression of pDNA powder compared with a control group with no siRNA. Naked siRNA inhalation powder with no vector showed the suppression of gene expression equivalent to that of an siRNA-polyethyleneimine complex without damaging tissues. These results show that the present method is suitable for evaluating the gene-silencing effect of inhaled siRNA powders.

Cationic polymers are being studied as non-viral gene delivery vectors. Poly{N-[N-(2-aminoethyl)-2-aminoethyl]aspartamide} (PAsp(DET)) and their block copolymers with poly(ethylene glycol), PEG-PAsp(DET), have been reported as efficient biodegradable non-viral vectors which form a polyplex with plasmid DNA (pDNA). However, the polyplexes are not stable because PAsp(DET) and PEG-PAsp(DET) are easily subjected to hydrolysis; therefore, they need to be prepared on site. In this study, using the biodegradable polycations as non-viral vectors, PAsp(DET) and PEG-PAsp(DET), we investigated the effects of L-leucine (Leu) on the polyplex. We prepared solutions and dry powders with and without Leu. Both dry powders had large and porous particles and Leu acted as a dispersing agent. The transfection activity of the sample solutions decreased within a month. However, the decrease in the transfection activity was partially suppressed by the dry powder with Leu at 5 and 25°C at 3 months. Furthermore, transfection experiments revealed that Leu exhibited a pDNA-stabilizing effect in the solution and dry powder. Similar results were observed for pDNA integrity, where a polyplex was formed in the dry powder. The results suggest that Leu is a candidate stabilizer to protect pDNA from degradation.

We evaluated the storage stability of powder containing naked plasmid DNA (pDNA) and hyaluronic acid (HA) or mannitol (Man) prepared by the spray-freeze-drying technique, through which we have reported high gene expression without any gene vectors. The powders composed of 5–10-µm porous particles and showing excellent dispersion were stored for 12 months under three storage conditions: 5 °C/Dry, 25 °C/Dry, and 25 °C/75% relative humidity. The humidified powders lost their porous shape within 1 week and were not suitable for inhalation characterization. On the other hand, the powders under dry conditions maintained high inhalation characteristics and pDNA integrity for 12 months. We administered the powders to the lungs of mice. The naked pDNA in HA powder showed significantly higher gene expression compared with that in Man powder and a pDNA-polyethylenimine complex solution. The gene expression of pDNA/HA powder was maintained for 12 months. These results suggest that powder containing naked pDNA is stable on storing under appropriate dry conditions and the naked pDNA/HA powder shows effective pulmonary gene expression.

In the development of drugs for intra-articular administration, sustained-release formulations are desirable because it is difficult to maintain the effect of conventional injections due to immediate drug leakage from the joint cavity. In this study, a sustained-release poly(lactic-co-glycolic acid) (PLGA) microsphere formulation for intra-articular administration containing indocyanine green (ICG) as a model drug was prepared to follow its fate after intra-articular administration in rats with a real-time in-vivo imaging system. ICG administered as an aqueous solution leaked from the joint cavity in a short time and was excreted outside the body within 1-3 d. However, ICG in the sustained-release formulation was retained in the joint cavity and released for 2 weeks. Next, a sustained-release formulation containing PLGA microspheres in a hyaluronic acid (HA) gel formulation was prepared. After gradual release in two stages, we could achieve sustained release for a longer period. It is considered that a combination formulation of PLGA microspheres and HA gel can significantly improve the sustained release of a drug administered into the knee joint.

In the present study, a novel dry small interfering RNA (siRNA) powder for inhalation, containing polyethyleneimine (PEI) as a delivery vector, was produced by spray freeze drying (SFD). The powder had spherical and highly porous structure of approximately 10 μm in diameter with high aerosol performance for emission and lung delivery. The reconstituted siRNA/PEI complex after dissolution of the powder had almost the same physicochemical properties and in vitro gene silencing activity as the original one constituted in the sample solution before SFD, showing that the integrity of the siRNA was maintained. In in vivo studies of intratracheal administration into lung metastasis mice and healthy mice, powder with a low dose of 3 μg siRNA exhibited strong and specific gene silencing activity against tumors metastasized to the lungs, whereas it caused no significant histological changes, lactate dehydrogenase leakage, or inflammatory cytokine induction in the lungs. These results strongly indicated that inhalable dry siRNA/PEI powders can provide effective pulmonary gene silencing without severe lung injury and that SFD can be applied to the production of such powders.

Extracellular histones promote platelet aggregation and thrombosis; this is followed by induction of coagulation disorder, which results in exhaustion of coagulation factors. Complement component 5 (C5) is known to be associated with platelet aggregation and coagulation system activation. To date, the pathological mechanism underlying liver injury has remained unclear. Here, we investigated whether C5 promotes liver injury associated with histone-induced lethal thrombosis. C5-sufficient and C5-deficient mice received single tail vein injections of purified, unfractionated histones obtained from calf thymus (45–75 μg/g). Subsequently, the mice were monitored for survival for up to 72 h. Based on the survival data, the 45 μg/g dose was used for analysis of blood cell count, liver function, blood coagulation ability, and promotion of platelet aggregation and platelet/leukocyte aggregate (PLA) production by extracellular histones. C5-deficient mice were protected from lethal thrombosis and had milder thrombocytopenia, consumptive coagulopathy, and liver injury with embolism and lower PLA production than C5-sufficient mice. These results indicate that C5 is associated with coagulation disorders, PLA production, and embolism-induced liver injury. In conclusion, C5 promotes liver injury associated with histone-induced lethal thrombosis.

In this study, a novel dry small interfering RNA (siRNA) powder for inhalation, containing chitosan and mannitol, was prepared using the supercritical carbon dioxide (CO2) technique. Although the siRNA/chitosan powder was difficult to disperse because of a long needle-like structure, it could be reduced to fragments of 10-20 µm by manual grinding, which allowed for administration into mice. Electrophoresis revealed that the supercritical CO2 technique and manual grinding didn't greatly affect the integrity of the siRNA. Furthermore, the siRNA was more stable in the lungs than in blood, suggesting the utility of pulmonary delivery. Biodistribution experiments using Cy5.5-labeled siRNA demonstrated that pulmonary administration of the powder achieved a prolonged exposure of the siRNA/chitosan complex on the lung epithelial surface at a higher concentration. For the evaluation of the in-vivo gene silencing effect of the siRNA/chitosan powder, mice bearing colon26/Luc cells were used. The powder significantly inhibited the increase in luminescence intensity in the lungs, but the siRNA/chitosan solution and a non-specific dry siRNA/chitosan powder didn't, indicating the effective and specific gene silencing against the tumor cells metastasized in the lungs of mice by the siRNA/chitosan powder. These results strongly indicate that inhalable dry siRNA powders have the possibility of effective pulmonary gene silencing and that the supercritical CO2 technique can be applied to the production.

Spray–freeze drying (SFD) is an attractive technique to prepare highly porous dry powders for inhalation. However, there have been few reports of its application to dry powder inhalers (DPIs). Therefore, in this study, we prepared dry plasmid DNA (pDNA) powders with different molecular ratios of chitosan to pDNA (N/P ratios) by SFD. All the pDNA powders were spherical and highly porous, with particles approximately 20–40 μm in geometric diameter. The morphology changed little with the alteration of the N/P ratio. On electrophoresis, a band of linear pDNA was detected in the preparation without chitosan, suggesting the destabilization of pDNA through SFD. However, the addition of chitosan protected pDNA from destabilization. Moreover, the pDNA powders were evaluated for pulmonary gene transfection efficiency using an in vivo dual imaging technique for gene DPIs developed previously. Maximum gene expression was observed at 9–12 h following pulmonary administration of the powders into mice. The powder with the N/P ratio of 10 had the highest gene transfection efficiency. A higher affinity of chitosan for pDNA and a smaller (approximately 100 nm) pDNA/chitosan complex (N/P f 10) were found at pH 6.5 (in lung) than at pH 7.4 (in physiological conditions), suggesting that the effective compaction of pDNA by chitosan at the N/P ratio of 10 at pH 6.5 contributes to the gene transfection efficiency in the lung. These results suggest inhalable dry pDNA powders with chitosan prepared by SFD to be a suitable formulation for pulmonary gene therapy.

Fenretinide (N-(4-hydroxyphenyl)retinamide, 4-HPR) is a synthetic retinoid with high antitumor activity against a variety of malignant cells in vitro, and is a promising candidate for cancer chemoprevention and chemotherapy. To enhance the antitumor efficacy of 4-HPR in vivo, 4-HPR were encapsulated into polymeric micelles for tumor targeting by enhanced permeability and retention effects. 4-HPR encapsulated in poly(ethylene glycol)-poly(benzyl aspartate) block copolymer micelles were prepared by the evaporation method. The mean particle size of 4-HPR encapsulated in polymeric micelles was about 173 nm. After intravenous injection into tumor-bearing mice, the delivery of 4-HPR by polymeric micelles increased the blood concentration and enhanced the tumor accumulation of 4-HPR over the injection of the 4-HPR encapsulated in oil-in-water (O/W) emulsions. Tumor growth was significantly delayed following treatment by 4-HPR encapsulated in polymeric micelles, which demonstrated the improved in vivo antitumor efficacy of 4-HPR. In addition, 4-HPR encapsulated in polymeric micelles did not cause any body weight loss. These results suggest that polymeric micelles are a promising and effective carrier of 4-HPR in order to enhance tumor delivery and have potential application in the treatment of solid tumor.

RNA interference (RNAi) is a technology that specifically inhibits gene expression and is carried out by small 21-27-nucleotide double-stranded small interfering RNA (siRNA) or short hairpin RNA (shRNA). RNAi has become a very promising technology for genetic research, however problems remain with the delivery of siRNA into cells. SiRNA and shRNA are easily degraded by RNases in body fluids and are hardly able to permeate cell membranes because of hydrophilic, polyanionic macromolecules which make their bioavailability very low. We have focused on the third double-stranded RNA-binding domain (dsRBD3) from the Mus musculus Staufen protein in order to develop a non-viral, multifunctional, artificial virus-like delivery system. We constructed a dsRBD3 expression vector and the recombinant dsRBD3 was expressed as a fusion protein with affinity tags. Purified dsRBD3 was mixed with siRNA or shRNA at various ratios and then added to fetal bovine serum (FBS) to evaluate the inhibition of the degradation of double-stranded RNA (dsRNA) by RNase. Unexpectedly, dsRBD3 was not able to protect the siRNA against degradation by FBS, but shRNA was stabilized to some degree by dsRBD3.

Dual imaging of lung deposition and gene expression following the pulmonary delivery of a gene formulation is useful for a precise analysis of gene transfection efficiency in vivo. As a novel probe for evaluating lung deposition, in this study, a poly(ethylene glycol)-conjugated near-infrared fluorescent probe (PEG-NIRF) was newly synthesized, and compared with indocyanine green (ICG), for application to pDNA/polyethyleneimine (PEI) complex. PEG-NIRF had superior characteristics including a larger Stokes shift (absorption maximum, 662 nm; emission maximum, 772 nm) and relatively equivalent fluorescence intensity compared with ICG. ICG affected the physicochemical properties of pDNA/PEI complex with a loss of fluorescence intensity, while PEG-NIRF did not. Experiments in mice demonstrated that PEG-NIRF showed greater lung localization than ICG following pulmonary co-delivery with pDNA/PEI complex, indicating the possibility of accurately evaluating lung deposition. Moreover, it was clarified that the evaluation of lung deposition by PEG-NIRF even at 60 min could be significantly correlated with gene expression in each mouse following pulmonary co-delivery with pDNA/PEI complex. These results suggest that PEG-NIRF is widely applicable to the dual imaging of the lung deposition and gene expression of inhaled gene formulations.

The development of efficient and selective therapeutic gene delivery methods is a potential medical treatment for intractable diseases. Dry powder inhalers (DPIs) can efficiently deliver drugs locally to the lung. Many reports discuss preparation methods for DPIs. Spray-freeze drying is a method by which highly porous particulates can be prepared. However, altered physical properties after preparation may result in changes in gene expression. In this study, bovine serum albumin (BSA) was added as a lyoprotectant, and 1,2-dioleoyl-3-trimethylammonium propane/cholesterol liposomes/pCMV-Luc DPIs (lipoplex DPIs) were prepared by spray-freeze drying. The mean particle sizes of the lipoplex DPIs prepared without BSA increased by approximately 6.7-fold compared with that of the lipoplexes solution. In contrast, the mean particle sizes of the lipoplex/BSA DPIs increased only slightly. Gene expression was evaluated after the intratracheal administration of the lipoplexes solution, with maximum gene expression observed at 12 h after the administration. In contrast, maximum gene expression of the lipoplex/BSA DPIs occurred at 6 h after administration. The gene expression associated with the lipoplex DPIs was significantly lower compared with that of the lipoplex/BSA DPIs at 6 (p

For stable encapsulation of N-(4-hydroxyphenyl)retinamide (4-HPR) into polymeric micelles, four types of block copolymers were synthesized with different esterified functional groups: heptyl (C7), nonyl (C9), benzyl (Bz), and phenylpropyl (C3Ph). The stability of 4-HPR encapsulated polymeric micelles was evaluated by measuring the blood concentration of 4-HPR in mice. After intravenous administration of 4-HPR and 4-HPR encapsulated PEG liposomes, the blood concentration of 4-HPR was about 2.8% and 2.2% of the dose/mL, suggesting the rapid release of 4-HPR from PEG liposomes. In contrast, the blood concentration of 4-HPR after intravenous administration of all 4-HPR encapsulated polymeric micelles studied was much higher (about 22-34% of the dose/mL). Among them, the polymeric micelles prepared by block copolymers (Bz) showed the highest blood concentration of 4-HPR. As far as the effects of the level of Bz groups in the block copolymers are concerned, the blood concentration of 4-HPR was enhanced by Bz groups at a level of 72% and 77%, but not by Bz groups at a level of 43% and 51%. These results suggest that 4-HPR is stably encapsulated in polymeric micelles prepared by block copolymers (Bz) but a level of over 72% of Bz groups is needed. These findings will be of value in the future use, design, and development of polymeric micelles for in vivo application of 4-HPR.