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Anti-inflammatory loaded poly-lactic glycolic acid nanoparticle formulations to enhance myocardial gene transfer: an in-vitro assessment of a drug/gene combination therapeutic approach for direct injection

Anti-inflammatory loaded poly-lactic glycolic acid nanoparticle formulations to enhance... Background: Cardiac gene therapy for heart disease is a major translational research area with potential, yet problems with safe and efficient gene transfer into cardiac muscle remain unresolved. Existing methodology to increase vector uptake include modifying the viral vector, non-viral particle encapsulation and or delivery with device systems. These advanced methods have made improvements, however fail to address the key problem of inflammation in the myocardium, which is known to reduce vector uptake and contribute to immunogenic adverse events. Here we propose an alternative method to co-deliver anti-inflammatory drugs in a controlled release polymer with gene product to improve therapeutic effects. Methods: A robust, double emulsion production process was developed to encapsulate drugs into nanoparticles. Briefly in this proof of concept study, aspirin and prednisolone anti-inflammatory drugs were encapsulated in various poly-lactic glycolic acid polymer (PLGA) formulations. The resultant particle systems were characterized, co-delivered with GFP plasmid, and evaluated in harvested myocytes in culture for uptake. Results: High quality nanoparticles were harvested from multiple production runs, with an average 64 ± 10 mg yield. Four distinct particle drug system combinations were characterized and evaluated in vitro: PLGA(50:50) Aspirin, PLGA (65:35) Prednisolone, PLGA(65:35) Aspirin and PLGA(50:50) Prednisolone Particles consisted of spherical shape with a narrow size distribution 265 ± 104 nm as found in scanning electron microscopy imaging. Prednisolone particles regardless of PLGA type were found on average ≈ 100 nm smaller than the aspirin types. All four groups demonstrated high zeta potential stability and re-constitution testing prior to in vitro. In vitro results demonstrated co uptake of GFP plasmid (green) and drug loaded particles (red) in culture with no incidence of toxicity. Conclusions: Nano formulated anti-inflammatories in combination with standalone gene product therapy may offer a clinical solution to maximize cardiac gene therapy product effects while minimizing the risk of the host response in the inflammatory myocardial environment. Keywords: Combination therapy, Cardiac gene delivery, Nanotechnology, Myocyte expression * Correspondence: [email protected] Thoracic and Cardiovascular Surgery, Sanger Heart & Vascular Institute, Carolinas Healthcare System, Charlotte, NC, USA Full list of author information is available at the end of the article © 2014 Fargnoli et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Fargnoli et al. Journal of Translational Medicine 2014, 12:171 Page 2 of 9 http://www.translational-medicine.com/content/12/1/171 Background limited distribution of gene therapeutic per delivery site Acquired heart disease from myocardial infarction (i.e. requiring many injections [21]. Increasing the number of heart attack) remains the leading cause of mortality and injections has the adverse effect of triggering inflamma- morbidity worldwide, with 22 million new patients diag- tion in the myocardium, thus limiting the availability of nosed annually. Essentially, all approved pharmacologic additional injection sites and jeopardizing the retained and device systems impose significant cost burden to the therapy. The immune response to gene therapy prod- health system, yet fail to increase survival rates [1-3]. ucts, especially notorious with the viral mediated products, Heart transplant, which is the gold standard for patients, is complex but several key studies have demonstrated a will never meet clinical demand due to the chronic short- clearer relationship between inflammation and the in- age of viable donors [4,5]. Therefore, new therapeutic creased risk of an adaptive immune response [22,23]. approaches to manage the disease burden represent a Therefore it is postulated the use of an anti-inflammatory significant unmet need. Recently, sophisticated molecular drug co-delivered with the gene therapy product could: profiling tools combined with a deeper knowledge base (1) Address the inflammation to minimize the adaptive derived from disease models have ushered in a new era of immune response and promote therapeutic tolerance (2) biopharmaceutical development for heart disease. This increase trafficking and uptake in a more favorable has resulted in the development of a more potent class of microenvironment and (3) potentially permit more therapies designed to act at the myocyte level, whereby injection sites. therapeutic action is achieved primarily through DNA, This concept of a direct injection drug/gene approach RNA and or microRNA genetic reprogramming [6]. Vari- has yet to be translated into the heart, whereby problems ous gene therapy concepts have been applied successfully exist with increasing uptake and extending the half-life in animal models demonstrating increased contractility, of anti-inflammatory drugs at the site of injection be- repaired myocardium, and or regenerated new vessels to yond the peak acute inflammatory window of 48 hours. reduce myocardial infarction reoccurrence [7-10]. Inde- In addition to the timing issue, the anti-inflammatory pendent of the targeted gene mechanism, the most load must not interfere with vector trafficking or the common means to achieve these aims are with either subsequent gene expression efficiency. Numerous stud- bioengineered viral or non-viral vector biologics, since ies have explored of advanced non-viral vectors to in- the uptake and success rates of naked molecular ther- crease in vivo performance by means of transfection apies is very poor in vivo [11,12]. The most effective alone [24,25]. However, none have attempted to use gene products today have shown remarkable promise, anti-inflammatories at the injection site co-delivered but at the same time have also presented more risks with a higher risk, but optimal gene transfer vector to and complicated translational issues, especially when provide a more promising clinical strategy. This study compared with traditional pharmaceutical compounds. summarizes the development and parameter testing of a Despite the availability of effective transgene-vector reliable nanoscale anti-inflammatory formulation pro- systems, one major rate limiting problem is with achiev- duction process for co-delivery with gene products. The ing safe and efficient myocardial gene transfer in the development phase features aspirin and prednisolone, clinic [13,14]. Due to size scale and more complex mem- two widely utilized anti-inflammatories and incorporates brane barriers, these issues do not emerge in smaller them into two common FDA approved poly lactic gly- animal studies yet are a major challenge in larger organ- colic acid (PLGA) polymers [26]. Complete nanoparticle isms [15,16]. Although the preferred route of adminis- characterization, process tolerance limits and an in vitro tration in clinical trials, it remains controversial whether feasibility assessment in harvested myocytes are offered to or not minimally invasive catheter infusion approaches evaluate the concept of a drug/gene combination therapy. can yield sufficient therapeutic expression levels that sig- nificantly improve outcomes in the clinic [17]. Another Methods major problem with these systems is restricting thera- Poly-lactic glycolic acid nanoparticle production process peutic expression to the heart and minimizing off target Pre-Processing Steps: effects. In fact, published large animal data has demon- strated a greater than 2000 fold higher presence in A water oil water (w/o/w) double emulsion process collateral organs versus the heart [18-20]. Alternatively, outlined in (Figure 1) was executed to generate aspirin direct myocardial delivery methods can restrict thera- (99% pure, Sigma Aldrich USA) and prednisolone (99% peutics to the heart if safely administered. pure, Sigma Aldrich USA) loaded poly- lactic glycolic acid Direct myocardial delivery methods (e.g. needle injec- (PLGA) nanoparticles (NPs). First, initial drug load water tion, sonoporation) can offer greater cardiac specificity phase stocks of 1–3 mg/mL aspirin and 0.1-0.4 mg/mL of gene therapeutics compared to percutaneous infusion prednisolone were created by dissolving in 1% poly vinyl approaches. The key unresolved problem is with the alcohol (PVA) solution. In the case of prednisolone due to Fargnoli et al. Journal of Translational Medicine 2014, 12:171 Page 3 of 9 http://www.translational-medicine.com/content/12/1/171 Figure 1 The water oil water double emulsion nanoparticle production process work flow sequence to generate high quality anti-inflammatory formulations. its poor water solubility, a 10% Ethanol (w/w%) was added. Multiple images were taken from separate locations on the These doses were selected based on body weight and field with focus in the range of 1 um to 500 nm. pharmacokinetic data for the rodent species. The second step or oil phase was generated in a separate vial, with Stability testing PLGA, input mass range (20–120 mg) of one of either 24 hour formulation stability test types (50:50, 65:35 i.e. % of lactic: glycolic acid chains) Saline stability re-constituted particles tests were also dissolved in 2.5 mL of Dichloromethane. For the in vitro conducted. Ten mg of freeze dried nanoparticles were study only, production runs were carried out as described dissolved and probe sonicated in sterile 0.9% saline water in the methods above except 100 μg of Rhodamin B dye and allowed to settle over a 24 hour period. Repeat powder was added to the first drug water phase. droplets were first dried and sputter coated for loading into the SEM. Process Steps: Zeta potential colloidal stability measurements The first emulsion was created by adding 1 mL of as- A sample of each particle composition was prepared in pirin or prednisolone drug PVA 1% solution dropwise to water and added to testing cuvettes per manufacturer in- the oil phase polymer in a 5 mL glass vial under probe structions of the Zetasizer Nano ZS instrument (Malvern sonication. After 3 minutes, this resultant emulsion was Instruments, UK). Triplicate runs were averaged to repre- then added dropwise to a larger outer water phase con- sent a single data point for multiple samples from the taining 15 mL of PVA 1% to create the double emulsion. same production lot. The double emulsion was then placed in a fume hood and stirred gently for at least 24 hours to facilitate Controlled release & loading efficiency analysis solvent evaporation and particle formation. Separation AUV–vis spectrophotometer (NanoDrop, National In- was achieved with via ultra-centrifugation at 30,000 g for struments) was used to generate two separate standard 35 min at 10C. The resultant particle pellets were washed curves for serial dilutions of known drug concentrations. to remove residual drug/polymer, then freeze dried over- The wavelength consistent with aspirin detection was night. Four nanoparticle compositions were generated 275 nm and 235 nm for prednisolone. with the reaction: PLGA (50:50 Aspirin), PLGA (65:35 Aspirin), PLGA (50:50 Prednisolone) and PLGA (65:35 Drug loading efficiency calculations Prednisolone). To compute loading efficiency, the amount of either as- pirin or prednisolone encapsulated in the nanoparticle Post-Processing: formulations was determined by measuring the residual amount in the supernatant following centrifugation rela- All yields were weighed then stored in sterile cryovial tive to the initial load in the water phase. Percentage containers at -20C. was derived as the mass amount of drug remaining in the supernatant following separation. Scanning electron microscopy (SEM) analysis & characterization Controlled release analysis Approximately 5 mg of each freeze dried NP sample was High quality yields were selected based on the most potent prepared for SEM with gold sputter coating, and then im- drug formulation of 5 wt. % PLGA aspirin and 1.5% PLGA aged on a JEOL SEM unit at 1.00 kV between 10–20,000 x. prednisolone were manufactured per process specifications. Fargnoli et al. Journal of Translational Medicine 2014, 12:171 Page 4 of 9 http://www.translational-medicine.com/content/12/1/171 Each particle formulation was prepared for controlled re- Complex 2- 8 μL of Lipofectamine (Invitrogen) was lease studies as follows: (1) 20 mg of particle was dissolved diluted into 100 μL of media. into 10 mL of 42C (2) Samples for spectrophotometry ana- Then, complexes 1 and 2 were mixed together and lysis were removed with a syringe 450 nm filter at 12 hours, permitted to incubate at room temperature for at least 1, 2, 3, 4, 5 days (3) The sample volume was replaced and 20 minutes. After 20 minutes the contents of the indi- the process repeated for each interval up until the final vidual eppendorf yields were then transferred into each point (4) Triplicate UV–vis spectrophotometry measure- well. The plate was gently rocked then placed back in ments against the standard curve for each drug were per- the incubator until the first 24 hour imaging time point. formed on each sample to determine the percentage For the nanoparticle treatment wells as designated, the released for each run. 500 μL filtered sterile solution was added via syringe to each well with a 450 nm to prevent aggregates from transferring. In vitro testing protocol Neo-natal rat cardiac-myocyte harvesting Day 0 to 3 neonatal pups are used and the pups were Follow up fluorescent microscopy euthanatized by decapitation and the heart was immedi- The first set of images was taken at 24 hours post trans- ately removed with forceps. The atria and great vessels fection. Media was removed from each well prior to im- were removed and the left ventricular tissue was minced aging and replaced prior to returning to the incubator. and subjected to a trypsin-based disaggregation proced- The remaining set of images was taken at the 48 hour ure in a 6 well plate with ethanol cleaned scissor, rinsed time point. with HBSS with 1% P/S/G, and place in a 50 ml conical Fluorescence images were acquired using a Nikon Eclipse tube containing 10 ml of Trypsin solution for shaking TE2000-U fluorescence microscope equipped with a Plan (200 rpm) at 37C for 15 min. Cells were then centri- Fluor × 20/0.50 objective (Nikon, Tokyo, Japan. Microscope fuged at 660 rpm at 4c for 5 minutes. The supernatant controlling and image processing were carried out using was discarded and the cells were re-suspended in 20 ml Image-Pro Plus 4.5.1.27 (Media Cybernetics, Bethesda, of media and pre-plate for 1–3 hours in the incubator. MD, USA). Harvested cells were collected with centrifuge spin at 660 rpm for 5 min at room temperature. Cell pellets for Statistical analysis experiments were then placed in the culture media and All SEM and nanoparticle characterization data was loaded counted using 0.4% Trypsin blue. into GraphPrism software suite for statistical testing. Single way ANOVA was utilized to determine differences in Plasmid GFP DNA & tagged nanoparticle preparation nanoparticle subtypes. Individual paired t-tests were A 10 mg master aliquot of eGFP plasmid DNA was ob- used to compare across individual groups. Bonferroni tained from Invitrogen and handled according to manu- corrections were applied for significance testing. facturer’s instructions. Under sterile conditions working yields for each well were created with, 6 μg of DNA was Results diluted into 100 μL of RNAase free water. Separately Process capability 10 mg of each of the process output 4 resultant particle Over 45 nanoparticle production yields were obtained systems [PLGA50:50-Aspirin, PLGA50:50-Prednisolone, over the development course with the optimal ranges. PLGA65:35-Aspirin, PLGA65:35 Prednisolone] tagged with The process volumes were held in a fixed ratio, featuring Rhodamin B was finely crushed and mixed into 20 mL of water phase #1 at 1 mL, the oil phase at 3 mL, and the phosphate buffer saline. To remove residual dye not bound outer water phase #2 at 15 mLs. Pilot runs in greater within the particle structure, the particle solution was placed amount adhering to this proportion scale yielded the into a dialysis membrane submerged in an outer bath of same quality particles. Briefly, basic guidelines for each PBS at 37. Prior to well transfection, 10 μg of each nanopar- process phase. ticle solution in 500 μL was placed in individual aliquots. Water Phase: Aspirin 1–3 mg dissolved in PVA 1% or Prednisolone 0.02-1 mg in 10% ethanol PVA1%. It was DNA and nanoparticle well transfection noted that adding additional solvents to increase drug On the day before transfection, cells were placed in 12 load in this phase resulted in failure to maintain particle well plates with each well seeded at a density of 500,000 integrity and stability. myocytes in 1 mL of DMEM (GIBCO) media without an- Oil Phase: The process was very flexible in terms of tibiotics. The transfection complexes were then prepared: changing the amount of polymer added to the system Complex 1 – One 6 μg of DNA aliquot was diluted and was stable in the range of 20 – 120 mg of either into 100 μL of media in an individual eppendorf vial. PLGA type. Fargnoli et al. Journal of Translational Medicine 2014, 12:171 Page 5 of 9 http://www.translational-medicine.com/content/12/1/171 Outer Water Phase: The PVA in the system acts as a narrow, of high quality and was as follows: PLGA50:50 vital stabilizer that can be readily increased. Increases Prednisolone [234 ± 9 nm], PLGA65:35 Prednisolone beyond 2% tended to inhibit the amount but not the [228 ± 7 nm], PLGA50:50 Aspirin [323 ± 13 nm] and quality of generated nanoparticles. Thus, a working PLGA65:35 Aspirin [302 ± 7 nm]. ANOVA indicated range of 0.5-2% of PVA stabilizer in the outer water significance between the groups, specifically it was phase is suitable for accommodating various drug/polymer determined that aspirin contributed to larger particles complexes with good stability. as both PLGA50:50 and PLGA 65:35 types were signifi- cantly larger than their matched prednisolone counter- Particle characterization parts. (Figure 2C) This difference in size was most The results presented here summarize the characterization likely attributable to both higher aspirin mass content for each of the 4 resultant nanoparticle types acquired from and charge of the first water phase in the reaction 5 consecutive runs. Polymer load was fixed at 60 mg, sincesize was unaffected by theadditionof morepoly- aspirin 3 mg and prednisolone at 1 mg respectively. mer (data not shown). SEM images from the various runs for each nanoparticle Yields were very consistent and proportional to polymer type were loaded into ImageJ software for analysis. The mass input in the range of 75-80% recovery upon final har- process consistently yields uniform, spherically shaped vest. The average yields per polymer/drug type based on formulations (Figure 2A,B). The size distribution was very 60 mg input were: PLGA50:50 Prednisolone [46 ± 1 mg], Figure 2 SEM Characterization Sizing Results. A. Narrow size distribution and high quality spherical shaped yield example in A at 2μm scaling. B. Close up 500 nm scaling image indicates narrow size distribution in the 200-350 nm range consistently for all manufactured yields. C. Nanoparticle average size by drug and polymer combination. Aspirin nanoparticles of either 50:50 or 65:35 type had greater size (p<0.05) versus prednisolone. Fargnoli et al. Journal of Translational Medicine 2014, 12:171 Page 6 of 9 http://www.translational-medicine.com/content/12/1/171 PLGA65:35 Prednisolone [45 ± 2 mg], PLGA50:50 Aspirin [48 ± 1 mg] and PLGA65:35 Aspirin [47 ± 2 mg]. Produc- tion yields with increased or decreased polymer loading revealed the same results (data not shown). Loading Efficiency results were uniform for all 4 nano- particle types, independent of drug or polymer and were: PLGA50:50 Prednisolone [88.9 ± 0.01%], PLGA65:35 Pred- nisolone [88.2 ± 0.01%], PLGA50:50 Aspirin [89.0 ± 0.01%] and PLGA65:35 Aspirin [88.8 ± 0.01%]. Stability analysis Figure 4 Controlled release study results demonstrate that Positive nanoparticle visualization was realized on the aspirin particles overall release faster than prednisolone types. SEM 24 hours after re-constituting freeze dried product in saline for all 4 polymer configurations. The particle shape and size was retained. limited by a number of key variables. Therefore, produc- The stability of the nanoparticles in suspension was tion with major deviations with the water phase I input moderate to good in the range of −30 to −53 mV. A (data not shown) resulted in lower quality profiles featur- score much less than −30 indicates a stability issue with ing aggregation and wider size ranges. The first major crit- a pharmaceutical dispersion, while any score higher ical variable was the concentration of the loading drug in than −60 indicates maximum. The potential scores by the first water phase, which was largely limited by the nanoparticle type shown in Figure 3 were as follows: inherent solubility at room temperature. In the case of as- PLGA50:50 Prednisolone [−47 ± 5 mV], PLGA65:35 pirin, without solvents added, the maximum concentration Prednisolone [−31 ± 1 mV], PLGA50:50 Aspirin [−45 ± was 3 mg/mL directly at the solubility limit. Runs at the 0.5 mV] and PLGA65:35 Aspirin [−32 ± 0.9 mV]. Stat- 5–10 mg/mL range resulted in aggregation and lower istical tests revealed that the PLGA50:50, independent quality. In the case of prednisolone, it was anticipated of drug load was superior compared with the 65:35 that on a per gram basis at least 1 mg/mL would be re- type. quired to achieve high quality in addition to a realistic dosing paradigm for a rodent heart with target of 1 Controlled release of the nanoparticle formulations gram mass. This was achieved suitably with 10% Etha- Figure 4 shows a graphical depiction of the release over nol, however concentrations greater than 25% in an the span of 5 days. It was evident that the aspirin attempt to load more drug distorted the process (data released faster overall as compared with prednisolone. not shown). The PLGA and PVA stabilizer system as This is most likely due to a combination of factors presented here is therefore open to excipient manipu- including size, stability and charge. The PLGA50:50 lation provided that solubility and other attributes of Aspirin type had the fastest release profile. the selected drug are addressed such to prevent devia- tions in overall quality which may or may not be de- Process limitations sired depending on the application. We anticipate this The high quality in terms of particle shape uniformity, platform would be open for further experimentation by yield, surface charge and release properties were critically professional formulation scientists tailored to each specific PLGA/drug selection for the intended direct injection application. In-vitro myocyte transfection All wells were checked for viability and it was deter- mined that none indicated any major media discolor- ation or visual evidence of contamination. The following 5 groups all had positive detection of GFP (green) in at least 2/3 replicate wells at both 24 and 48 hours, with a greater degree of cells positive as expected at 48 hours. Figure 5 depicts independent uptake of both GFP plas- mid and nanoparticle co-signal. The absorption clusters Figure 3 Nanoparticle zeta potential colloidal stability testing were confirmed in the center of myocytes. The multiple results indicate that the PLGA50:50 nanoparticles are more DNA and nanoparticle infection groups yielded nano- stable in solution versus the PLGA65:35 types. particle presence (red) or both (yellow) at the 24 and 48 Fargnoli et al. Journal of Translational Medicine 2014, 12:171 Page 7 of 9 http://www.translational-medicine.com/content/12/1/171 regions, which are characterized by a high degree of inflammation and fibrosis. The second key finding in the final test was that PLGA uptake and release of anti- inflammatory agents in myocytes does not interfere with the absorption and trafficking of the GFP plasmid. Muscle tissue has a high risk of developing an adaptive immune response to gene products. Wilson et al. described in detail the host response after AAV delivery by route of administration and more specifically the role of inflam- mation [27]. A key finding with AAV mediated gene transfer was that the host either induces tolerance or an adaptive immune reaction through a series of com- plex interactions [28-30]. A prime risk factor in these interactions that was found to trigger adaptive immune responses were inflammatory cytokines and signals ei- ther already present in tissue or induced at the time of delivery [31]. It has been postulated that with attenu- ation of innate inflammatory response signals, the im- mune system has a much lower risk for mounting maladaptive T cell responses. Using the example of Figure 5 In Vitro Fluorescent Imaging at 48 hours post AAV, once vector capsid antigens are cleared from the transfection. All 4 particle systems exhibited safe and robust uptake in myocytes while not interfering with plasmid uptake and system, typically 12–16 weeks after delivery, there is a subsequent GFP expression. Yellow signal indicates co-existence of good chance for therapeutic tolerance. The risk is that GFP and nanoparticle in: A. PLGA65:35 Aspirin B. PLGA50:50 Aspirin an adaptive immune response will destroy those cells C. PLGA50:50 Prednisolone D. PLGA65:35 Prednisolone. expressing the transgene of interest well before these antigens are cleared. Use of anti-inflammatory agents to mitigate theinnateresponsetoinjuryislikelytore- post transfection: I. Control GFP DNA only (data not sult in enhanced long term gene expression. Intraven- shown) II. GFP and PLGA65:35 Aspirin (Figure 5A) ous delivery approaches are associated with a lower III. GFP and PLGA50:50 Aspirin (Figure 5B) IV. GFP level of induced inflammation but are also very ineffi- and PLGA50:50 Prednisolone (Figure 5C) V. GFP and cient. In contrast, the IM route in the heart remains at- PLGA65:35 Prednisolone (Figure 5D). tractive because greater cardiac specificity can be achieved, especially for angiogenesis or regenerative therapies that Discussion require a more local delivery profile. Yet IM delivery is This study presents two key findings that have broad im- associated with a more robust innate immune response plications for the advancement of cardiac gene therapies. due to associated tissue injury. First a reproducible, simple to use lab scale process was Direct injection into healthy or ischemic myocardial developed to generate anti-inflammatory nanoparticles regions introduces the gene product into a highly in- of very high quality for co-administration with gene flammatory region, which likely explains the poor car- products in a - regulatory friendly - PLGA platform. Al- diac gene therapy results with IM interventions. Early though only two anti-inflammatory drugs were utilized studies by Snyder et al. [32] reported that very little suc- in this feasibility assessment, it is anticipated that any cessful transfer occurs in damaged muscle in the inflam- matory environment. Numerous examples have validated other drug indicated for injection into muscle could be introduced by modification of the first drug water phase. these observations in gene therapy trials. In hemophilia Also the process offers an easy means to adjust the poly- trials for example it was found that IM injection into skeletal muscle resulted in transient therapeutic gene mer content in the oil phase for the desired degrad- ation/release profile, along with increasing the amount expression and an adaptive CD4+ immune response of stabilizer. Therefore this system can provide a plat- [33,34]. However, delivery of the same product infused into the liver has resulted in better outcomes and lim- form to guide future pre-clinical studies to investigate reliable clinical interventions to address the role of in- ited reactions. Muscular dystrophy trials have encoun- flammation on the relative performance of gene products tered similar difficulties and have attempted to utilize immunosuppressant drugs and other agents to limit in muscle tissue. The potential role of inflammation should not be overlooked, particularly in myocardial tissue responses after multiple IM injections compromising where the most common delivery scenario is in ischemic patient safety [35]. Fargnoli et al. Journal of Translational Medicine 2014, 12:171 Page 8 of 9 http://www.translational-medicine.com/content/12/1/171 Conclusions References 1. Gheorghiade M, Bonow RO: Chronic heart failure in the United States: a In this proof of concept study, GFP plasmid was utilized manifestation of coronary artery disease. Circulation 1998, 97:282–289. to simulate a therapeutic construct understanding that 2. Frazier OH, Myers TJ: Surgical therapy for severe heart failure. Curr Probl Cardiol naked DNA is likely to be at the lower end in terms of 1998, 23:721–768. 3. Delgado HD, Rao V, Ross JR, Verma S, Smedira N: Mechanical circulatory transduction efficiency. For more practical gene therapy assistance (State of Art). Circulation 2002, 106:2046–2050. applications, it is anticipated that viral vectors encoding 4. Siegenthaler PM, Martin J, Loo A, Doenst T, Bothe W, Beyersdorf F: the gene of interest could readily be combined with par- Implantation of the permanent jarvik-2000 left ventricular assist device. JACC 2002, 39:1764–1772. ticles containing potent anti-inflammatory drugs. The 5. Rose EA, Gelijns AC, Moskowitz AJ, Heitjan DF, Stevenson LW, Dembitsky W, hypothesis offered is that with the right formulation, the Long JW, Ascheim DD, Tierney AR, Levitan RG, Levitan RG, Watson JT, anti-inflammatory agent would be released at the suffi- Ronan NS, Meier P: Long-term use of a left ventricular assist device for end-stage heart failure. N Engl J Med 2001, 345:1435–1443. cient level over the critical post-delivery inflammatory 6. Schmia MC, Gumpert AM, Kock WJ: Cardiovascular gene therapy for period to provide an optimal viral vector trafficking myocardial infarction. Expert Opin Biol Ther 2014, 14:183–195. microenvironment. There would be a predicted increase 7. Kawase Y, Ly HQ, Prunier F, Lebeche D, Shi Y, Jin H, Hadri L, Yoneyama R, Hoshino K, Takewa Y, Sakata S, Peluso R, Zsebo K, Gwathmey JK, Tardif JC, in transduction efficiency, minimize the innate and adap- Tanguay JF, Hajjar RJ: Reversal of cardiac dysfunction after long-term tive immune response to the vector and/or transgene and expression of SERCA2a by gene transfer in a pre-clinical model of promote long term gene expression in the target tissues. heart failure. J Am Coll Cardiol 2008, 51(11):1112–1119. 8. Rengo G, Lymperopoulous A, Zincarelli C, Donniacuo M, Soltys S, This strategy of course would not be without its own limi- Rabinowitz JE, Koch WJ: Myocardial adeno-associated serotype 6 BARKct tations and would require much more experimentation gene therapy improves cardiac function and normalizes the neurhormonal to determine the best matched drug and release profile axis in chronic heart failure. Circulation 2009, 119(1):89–98. 9. Giacca M, Zacchigna S: VEGF gene therapy:therapeutic angiogenesis in for co-administration into the heart. More complex the clinic and beyond. Gene Ther 2012, 19(6):622–629. approaches in managing the host response following 10. Eulalio A, Mano M, Dal Ferro M, Zentilin L, Sinagra G, Zacchigna S, Giacca M: therapy have been applied, however it may turn out Functional screening identifies miRNAs inducing cardiac regeneration. Nature 2012, 492(7249):376–381. that simply addressing the innate immune response at 11. Wang W, Li W, Ma N, Steinhoff G: Non-viral gene delivery methods. the time of delivery may be a meritorious approach to Curr Pharm Biotechnology 2013, 14(1):46–60. advance successful clinical translation. 12. Su CH, Wu YJ, Wang HH, Yeh HI: Non-viral gene therapy targeting the cardiovascular system. Am J Physiol Heart Circ 2012, 303(6):629–638. 13. Katz MG, Swain JD, Tomasulo CE, Sumaroka M, Fargnoli A, Bridges CR: Competing interests Current Strategies for Myocardial Gene Delivery. J Mol Cell Cardiol 2011, The authors declare that they have no competing interests. 50(5):766–776. Epub 2010 Sep 15. 14. Katz MG, Fargnoli AS, Williams RD, Bridges CR: The road ahead: working towards effective clinical translation of myocardial gene therapies. Authors’ contributions Ther Deliv 2014, 5(1):39–51. doi: 10.4155/tde.13.134. ASF: Process development, particle evaluation, executed in vitro testing, 15. Logeart D, Hatem SN, Heimburger M, Le Roux A, Michel JB, Mercadier JJ: wrote manuscript. AM: Performed myocyte harvest and quality control; study How to optimize in vivo gene transfer to cardiac myocytes: mechanical design input. MGK: Study design input, manuscript drafting and revisions. or pharmacological procedures? Hum Gene Ther 2001, 12:1601–1610. RDW: Quantitative sizing analysis, figure generation, manuscript editing. KBM: 16. Katz MG, Fargnoli AS, Williams RD, Bridges CR: Gene therapy for enhancing Study design input, myocyte evaluation, manuscript editing. DBW: GPF viral and nonviral vectors for cardiac diseases: Current concepts and plasmid design and evaluation of results, manuscript editing. SY: future applications. Hum Gene Ther 2013, 24(11):914–927. Nanotechnology evaluations, experimental design, process development 17. Jessup M, Greenberg B, Mancini D, Cappola T, Pauly DF, Jaski B, Yaroshinsky A, techniques, manuscript editing. CRB: Study design and manuscript editing. Zsebo KM, Dittrich H, Hajjar RJ: Calcium Upregulation by Percutaneous All authors read and approved the final manuscript. Administration of Gene Therapy in Cardiac Disease (CUPID): a phase 2 trial of intracoronary gene therapy of sarcoplasmic reticulum Ca2+-ATPase in patients with advanced heart failure. Circulation 2014, 124(3):304–313. Acknowledgements doi: 10.1161/CIRCULATIONAHA.111.022889. Epub 2011 Jun 27. The preparation of this article was supported by the NIH grant 18. Byrne MJ, Power JM, Preovolos A, Mariani JA, Hajjar RJ, Kaye DM: 2R01HL083078-05, and partially by the James H. Heinemann Foundation. The Recirculating cardiac delivery of AAV2/1SERCA2a improves myocardial authors acknowledge the National Heart, Lung, and Blood Institute Gene function in an experimental model of heart failure in large animals. Therapy Resource Program. Marina Sumaroka PhD and Vladimir Shuvaev PhD Gene Ther 2008, 15(23):1550–1557. at the University of Pennsylvania provided assistance and consultation with 19. Kaye DM, Preovolos A, Marshall T, Byrne M, Hoshijima M, Hajjar R, Mariani JA, the microscopy evaluations. Jamie Ford PhD in the Singh Nanotechnology Pepe S, Chien KR, Power JM: Percutaneous cardiac recirculation-mediated Center at the University of Pennsylvania, provided guidance with the gene transfer of an inhibitory phospholamban peptide reverses advanced scanning electron microscopy applications. heart failure in large animals. J Am Coll Cardiol 2007, 50(3):253–260. 20. Bridges CR: 'Recirculating cardiac delivery' method of gene delivery Author details should be called 'non-recirculating' method. Gene Ther 2009, 16(7):939–940. Thoracic and Cardiovascular Surgery, Sanger Heart & Vascular Institute, Epub 2009 Apr 2. Carolinas Healthcare System, Charlotte, NC, USA. Internal Medicine, Perlman 21. Grossman PM, Han Z, Palasis M, Barry JJ, Lederman RJ: Incomplete retention School of Medicine, University of Pennsylvania Medical Center, Philadelphia, after direct myocardial injection. Cardiovasc Intervent 2002, 55(3):392–397. PA, USA. Pathology and Laboratory Medicine, Perlman School of Medicine, 22. Mingozzi F, Maus MV, Hui DJ, Sabatino DE, Murphy SL, Rasko JE, Ragni MV, University of Pennsylvania Medical Center, Philadelphia, PA, USA. Materials Manno CS, Sommer J, Jiang H, Pierce GF, Ertl HC, High KA: CD8(+) T-cell Science Engineering, School of Engineering & Applied Science, University of responses to adeno-associated virus capsid in humans. Nat Med 2007, Pennsylvania, Philadelphia, PA, USA. 13:419–422. Received: 31 March 2014 Accepted: 10 June 2014 23. Gao G, Wang Q, Calcedo R, Mays L, Bell P, Wang L, Vandenberghe LH, Published: 16 June 2014 Grant R, Sanmiguel J, Furth EE, Wilson JM: Adeno-associated virus-mediated Fargnoli et al. Journal of Translational Medicine 2014, 12:171 Page 9 of 9 http://www.translational-medicine.com/content/12/1/171 gene transfer to nonhuman primate liver can elicit destructive transgene-specific T cell responses. Hum Gene Ther 2009, 20(9):930–942. doi: 10.1089/hum.2009.060. 24. Yockman JW, Kastenmeier A, Erickson HM, Brumbach JG, Whitten MG, Albanil A, Li DY, Kim SW, Bull DA: Novel polymer carriers and gene constructs for treatment of myocardial ischemia and infarction. J Control Release 2008, 132(3):260–266. 25. Luo K, He B, Wu Y, Shen Y, Gu Z: Functional and biodegradable dendritic macromolecules with controlled architectures as nontoxic and efficient nanoscale gene vectors. Biotechnol Adv 2014, 32(4):818–830. doi: 10.1016/j. biotechadv.2013.12.008. Epub 2014 Jan 3. 26. Makadia HK, Siegel SJ: Poly Lactic-co-Glycolic Acid (PLGA) as Biodegradable Controlled Drug Delivery Carrier. Polymers (Basel) 2011, 3(3):1377–1397. 27. Mays LE, Wilson JM: The complex and evolving story of T cell activation to AAV vector-encoded transgene products. Mol Ther 2011, 19(1):16–27. Epub 2010 Nov 30. 28. Jooss K, Yang Y, Fisher KJ, Wilson JM: Transduction of dendritic cells byDNA viral vectors directs the immune response to transgene products in muscle fibers. J Virol 1998, 72:4212–4223. 29. Zhang Y, Chirmule N, Gao G, Wilson J: CD40 ligand-dependent activation of cytotoxic T lymphocytes by adeno-associated virus vectors in vivo: role of immature dendritic cells. J Virol 2000, 74:8003–8010. 30. Wang L, Dobrzynski E, Schlachterman A, Cao O, Herzog RW: Systemicprotein delivery by muscle-gene transfer is limited by a local immune response. Blood 2005, 105:4226–4234. 31. Yuasa K, Yoshimura M, Urasawa N, Ohshima S, Howell JM, Nakamura A, Hijikata T, Miyagoe-Suzuki Y, Takeda S: Injection of a recombinant AAV serotype 2 into canine skeletal muscles evokes strong immune responses against transgene products. Gene Ther 2007, 14(17):1249–1260. Epub 2007 Jun 21. 32. Snyder RO, Spratt SK, Lagarde C, Bohl D, Kaspar B, Sloan B, Cohen LK, Danos O: Efficient and stable adeno-associated virus-mediated transduction in the skeletal muscle of adult immunocompetent mice. Hum Gene Ther 1997, 8:1891–1900. 33. Wang L, Cao O, Swalm B, Dobrzynski E, Mingozzi F, Herzog RW: Major role of local immune responses in antibody formation to factor IX in AAV gene transfer. Gene Ther 2005, 12:1453–1464. 34. Cao O, Hoffman BE, Moghimi B, Nayak S, Cooper M, Zhou S, Ertl HC, High KA, Herzog RW: Impact of the underlying mutation and the route of vector administration on immune responses to factor IX in gene therapy for hemophilia B. Mol Ther 2009, 17:1733–1742. 35. Wang Z, Kuhr CS, Allen JM, Blankinship M, Gregorevic P, Chamberlain JS, Tapscott SJ, Storb R: Sustained AAV-mediated dystrophin expression in a canine model of Duchenne muscular dystrophy with a brief course of immunosuppression. Mol Ther 2007, 15(6):1160–1166. Epub 2007 Apr 10. doi:10.1186/1479-5876-12-171 Cite this article as: Fargnoli et al.: Anti-inflammatory loaded poly-lactic glycolic acid nanoparticle formulations to enhance myocardial gene transfer: an in-vitro assessment of a drug/gene combination therapeutic approach for direct injection. Journal of Translational Medicine 2014 12:171. Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Translational Medicine Springer Journals

Anti-inflammatory loaded poly-lactic glycolic acid nanoparticle formulations to enhance myocardial gene transfer: an in-vitro assessment of a drug/gene combination therapeutic approach for direct injection

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Springer Journals
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Copyright © 2014 by Fargnoli et al.; licensee BioMed Central Ltd.
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Biomedicine; Biomedicine general; Medicine/Public Health, general
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Abstract

Background: Cardiac gene therapy for heart disease is a major translational research area with potential, yet problems with safe and efficient gene transfer into cardiac muscle remain unresolved. Existing methodology to increase vector uptake include modifying the viral vector, non-viral particle encapsulation and or delivery with device systems. These advanced methods have made improvements, however fail to address the key problem of inflammation in the myocardium, which is known to reduce vector uptake and contribute to immunogenic adverse events. Here we propose an alternative method to co-deliver anti-inflammatory drugs in a controlled release polymer with gene product to improve therapeutic effects. Methods: A robust, double emulsion production process was developed to encapsulate drugs into nanoparticles. Briefly in this proof of concept study, aspirin and prednisolone anti-inflammatory drugs were encapsulated in various poly-lactic glycolic acid polymer (PLGA) formulations. The resultant particle systems were characterized, co-delivered with GFP plasmid, and evaluated in harvested myocytes in culture for uptake. Results: High quality nanoparticles were harvested from multiple production runs, with an average 64 ± 10 mg yield. Four distinct particle drug system combinations were characterized and evaluated in vitro: PLGA(50:50) Aspirin, PLGA (65:35) Prednisolone, PLGA(65:35) Aspirin and PLGA(50:50) Prednisolone Particles consisted of spherical shape with a narrow size distribution 265 ± 104 nm as found in scanning electron microscopy imaging. Prednisolone particles regardless of PLGA type were found on average ≈ 100 nm smaller than the aspirin types. All four groups demonstrated high zeta potential stability and re-constitution testing prior to in vitro. In vitro results demonstrated co uptake of GFP plasmid (green) and drug loaded particles (red) in culture with no incidence of toxicity. Conclusions: Nano formulated anti-inflammatories in combination with standalone gene product therapy may offer a clinical solution to maximize cardiac gene therapy product effects while minimizing the risk of the host response in the inflammatory myocardial environment. Keywords: Combination therapy, Cardiac gene delivery, Nanotechnology, Myocyte expression * Correspondence: [email protected] Thoracic and Cardiovascular Surgery, Sanger Heart & Vascular Institute, Carolinas Healthcare System, Charlotte, NC, USA Full list of author information is available at the end of the article © 2014 Fargnoli et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Fargnoli et al. Journal of Translational Medicine 2014, 12:171 Page 2 of 9 http://www.translational-medicine.com/content/12/1/171 Background limited distribution of gene therapeutic per delivery site Acquired heart disease from myocardial infarction (i.e. requiring many injections [21]. Increasing the number of heart attack) remains the leading cause of mortality and injections has the adverse effect of triggering inflamma- morbidity worldwide, with 22 million new patients diag- tion in the myocardium, thus limiting the availability of nosed annually. Essentially, all approved pharmacologic additional injection sites and jeopardizing the retained and device systems impose significant cost burden to the therapy. The immune response to gene therapy prod- health system, yet fail to increase survival rates [1-3]. ucts, especially notorious with the viral mediated products, Heart transplant, which is the gold standard for patients, is complex but several key studies have demonstrated a will never meet clinical demand due to the chronic short- clearer relationship between inflammation and the in- age of viable donors [4,5]. Therefore, new therapeutic creased risk of an adaptive immune response [22,23]. approaches to manage the disease burden represent a Therefore it is postulated the use of an anti-inflammatory significant unmet need. Recently, sophisticated molecular drug co-delivered with the gene therapy product could: profiling tools combined with a deeper knowledge base (1) Address the inflammation to minimize the adaptive derived from disease models have ushered in a new era of immune response and promote therapeutic tolerance (2) biopharmaceutical development for heart disease. This increase trafficking and uptake in a more favorable has resulted in the development of a more potent class of microenvironment and (3) potentially permit more therapies designed to act at the myocyte level, whereby injection sites. therapeutic action is achieved primarily through DNA, This concept of a direct injection drug/gene approach RNA and or microRNA genetic reprogramming [6]. Vari- has yet to be translated into the heart, whereby problems ous gene therapy concepts have been applied successfully exist with increasing uptake and extending the half-life in animal models demonstrating increased contractility, of anti-inflammatory drugs at the site of injection be- repaired myocardium, and or regenerated new vessels to yond the peak acute inflammatory window of 48 hours. reduce myocardial infarction reoccurrence [7-10]. Inde- In addition to the timing issue, the anti-inflammatory pendent of the targeted gene mechanism, the most load must not interfere with vector trafficking or the common means to achieve these aims are with either subsequent gene expression efficiency. Numerous stud- bioengineered viral or non-viral vector biologics, since ies have explored of advanced non-viral vectors to in- the uptake and success rates of naked molecular ther- crease in vivo performance by means of transfection apies is very poor in vivo [11,12]. The most effective alone [24,25]. However, none have attempted to use gene products today have shown remarkable promise, anti-inflammatories at the injection site co-delivered but at the same time have also presented more risks with a higher risk, but optimal gene transfer vector to and complicated translational issues, especially when provide a more promising clinical strategy. This study compared with traditional pharmaceutical compounds. summarizes the development and parameter testing of a Despite the availability of effective transgene-vector reliable nanoscale anti-inflammatory formulation pro- systems, one major rate limiting problem is with achiev- duction process for co-delivery with gene products. The ing safe and efficient myocardial gene transfer in the development phase features aspirin and prednisolone, clinic [13,14]. Due to size scale and more complex mem- two widely utilized anti-inflammatories and incorporates brane barriers, these issues do not emerge in smaller them into two common FDA approved poly lactic gly- animal studies yet are a major challenge in larger organ- colic acid (PLGA) polymers [26]. Complete nanoparticle isms [15,16]. Although the preferred route of adminis- characterization, process tolerance limits and an in vitro tration in clinical trials, it remains controversial whether feasibility assessment in harvested myocytes are offered to or not minimally invasive catheter infusion approaches evaluate the concept of a drug/gene combination therapy. can yield sufficient therapeutic expression levels that sig- nificantly improve outcomes in the clinic [17]. Another Methods major problem with these systems is restricting thera- Poly-lactic glycolic acid nanoparticle production process peutic expression to the heart and minimizing off target Pre-Processing Steps: effects. In fact, published large animal data has demon- strated a greater than 2000 fold higher presence in A water oil water (w/o/w) double emulsion process collateral organs versus the heart [18-20]. Alternatively, outlined in (Figure 1) was executed to generate aspirin direct myocardial delivery methods can restrict thera- (99% pure, Sigma Aldrich USA) and prednisolone (99% peutics to the heart if safely administered. pure, Sigma Aldrich USA) loaded poly- lactic glycolic acid Direct myocardial delivery methods (e.g. needle injec- (PLGA) nanoparticles (NPs). First, initial drug load water tion, sonoporation) can offer greater cardiac specificity phase stocks of 1–3 mg/mL aspirin and 0.1-0.4 mg/mL of gene therapeutics compared to percutaneous infusion prednisolone were created by dissolving in 1% poly vinyl approaches. The key unresolved problem is with the alcohol (PVA) solution. In the case of prednisolone due to Fargnoli et al. Journal of Translational Medicine 2014, 12:171 Page 3 of 9 http://www.translational-medicine.com/content/12/1/171 Figure 1 The water oil water double emulsion nanoparticle production process work flow sequence to generate high quality anti-inflammatory formulations. its poor water solubility, a 10% Ethanol (w/w%) was added. Multiple images were taken from separate locations on the These doses were selected based on body weight and field with focus in the range of 1 um to 500 nm. pharmacokinetic data for the rodent species. The second step or oil phase was generated in a separate vial, with Stability testing PLGA, input mass range (20–120 mg) of one of either 24 hour formulation stability test types (50:50, 65:35 i.e. % of lactic: glycolic acid chains) Saline stability re-constituted particles tests were also dissolved in 2.5 mL of Dichloromethane. For the in vitro conducted. Ten mg of freeze dried nanoparticles were study only, production runs were carried out as described dissolved and probe sonicated in sterile 0.9% saline water in the methods above except 100 μg of Rhodamin B dye and allowed to settle over a 24 hour period. Repeat powder was added to the first drug water phase. droplets were first dried and sputter coated for loading into the SEM. Process Steps: Zeta potential colloidal stability measurements The first emulsion was created by adding 1 mL of as- A sample of each particle composition was prepared in pirin or prednisolone drug PVA 1% solution dropwise to water and added to testing cuvettes per manufacturer in- the oil phase polymer in a 5 mL glass vial under probe structions of the Zetasizer Nano ZS instrument (Malvern sonication. After 3 minutes, this resultant emulsion was Instruments, UK). Triplicate runs were averaged to repre- then added dropwise to a larger outer water phase con- sent a single data point for multiple samples from the taining 15 mL of PVA 1% to create the double emulsion. same production lot. The double emulsion was then placed in a fume hood and stirred gently for at least 24 hours to facilitate Controlled release & loading efficiency analysis solvent evaporation and particle formation. Separation AUV–vis spectrophotometer (NanoDrop, National In- was achieved with via ultra-centrifugation at 30,000 g for struments) was used to generate two separate standard 35 min at 10C. The resultant particle pellets were washed curves for serial dilutions of known drug concentrations. to remove residual drug/polymer, then freeze dried over- The wavelength consistent with aspirin detection was night. Four nanoparticle compositions were generated 275 nm and 235 nm for prednisolone. with the reaction: PLGA (50:50 Aspirin), PLGA (65:35 Aspirin), PLGA (50:50 Prednisolone) and PLGA (65:35 Drug loading efficiency calculations Prednisolone). To compute loading efficiency, the amount of either as- pirin or prednisolone encapsulated in the nanoparticle Post-Processing: formulations was determined by measuring the residual amount in the supernatant following centrifugation rela- All yields were weighed then stored in sterile cryovial tive to the initial load in the water phase. Percentage containers at -20C. was derived as the mass amount of drug remaining in the supernatant following separation. Scanning electron microscopy (SEM) analysis & characterization Controlled release analysis Approximately 5 mg of each freeze dried NP sample was High quality yields were selected based on the most potent prepared for SEM with gold sputter coating, and then im- drug formulation of 5 wt. % PLGA aspirin and 1.5% PLGA aged on a JEOL SEM unit at 1.00 kV between 10–20,000 x. prednisolone were manufactured per process specifications. Fargnoli et al. Journal of Translational Medicine 2014, 12:171 Page 4 of 9 http://www.translational-medicine.com/content/12/1/171 Each particle formulation was prepared for controlled re- Complex 2- 8 μL of Lipofectamine (Invitrogen) was lease studies as follows: (1) 20 mg of particle was dissolved diluted into 100 μL of media. into 10 mL of 42C (2) Samples for spectrophotometry ana- Then, complexes 1 and 2 were mixed together and lysis were removed with a syringe 450 nm filter at 12 hours, permitted to incubate at room temperature for at least 1, 2, 3, 4, 5 days (3) The sample volume was replaced and 20 minutes. After 20 minutes the contents of the indi- the process repeated for each interval up until the final vidual eppendorf yields were then transferred into each point (4) Triplicate UV–vis spectrophotometry measure- well. The plate was gently rocked then placed back in ments against the standard curve for each drug were per- the incubator until the first 24 hour imaging time point. formed on each sample to determine the percentage For the nanoparticle treatment wells as designated, the released for each run. 500 μL filtered sterile solution was added via syringe to each well with a 450 nm to prevent aggregates from transferring. In vitro testing protocol Neo-natal rat cardiac-myocyte harvesting Day 0 to 3 neonatal pups are used and the pups were Follow up fluorescent microscopy euthanatized by decapitation and the heart was immedi- The first set of images was taken at 24 hours post trans- ately removed with forceps. The atria and great vessels fection. Media was removed from each well prior to im- were removed and the left ventricular tissue was minced aging and replaced prior to returning to the incubator. and subjected to a trypsin-based disaggregation proced- The remaining set of images was taken at the 48 hour ure in a 6 well plate with ethanol cleaned scissor, rinsed time point. with HBSS with 1% P/S/G, and place in a 50 ml conical Fluorescence images were acquired using a Nikon Eclipse tube containing 10 ml of Trypsin solution for shaking TE2000-U fluorescence microscope equipped with a Plan (200 rpm) at 37C for 15 min. Cells were then centri- Fluor × 20/0.50 objective (Nikon, Tokyo, Japan. Microscope fuged at 660 rpm at 4c for 5 minutes. The supernatant controlling and image processing were carried out using was discarded and the cells were re-suspended in 20 ml Image-Pro Plus 4.5.1.27 (Media Cybernetics, Bethesda, of media and pre-plate for 1–3 hours in the incubator. MD, USA). Harvested cells were collected with centrifuge spin at 660 rpm for 5 min at room temperature. Cell pellets for Statistical analysis experiments were then placed in the culture media and All SEM and nanoparticle characterization data was loaded counted using 0.4% Trypsin blue. into GraphPrism software suite for statistical testing. Single way ANOVA was utilized to determine differences in Plasmid GFP DNA & tagged nanoparticle preparation nanoparticle subtypes. Individual paired t-tests were A 10 mg master aliquot of eGFP plasmid DNA was ob- used to compare across individual groups. Bonferroni tained from Invitrogen and handled according to manu- corrections were applied for significance testing. facturer’s instructions. Under sterile conditions working yields for each well were created with, 6 μg of DNA was Results diluted into 100 μL of RNAase free water. Separately Process capability 10 mg of each of the process output 4 resultant particle Over 45 nanoparticle production yields were obtained systems [PLGA50:50-Aspirin, PLGA50:50-Prednisolone, over the development course with the optimal ranges. PLGA65:35-Aspirin, PLGA65:35 Prednisolone] tagged with The process volumes were held in a fixed ratio, featuring Rhodamin B was finely crushed and mixed into 20 mL of water phase #1 at 1 mL, the oil phase at 3 mL, and the phosphate buffer saline. To remove residual dye not bound outer water phase #2 at 15 mLs. Pilot runs in greater within the particle structure, the particle solution was placed amount adhering to this proportion scale yielded the into a dialysis membrane submerged in an outer bath of same quality particles. Briefly, basic guidelines for each PBS at 37. Prior to well transfection, 10 μg of each nanopar- process phase. ticle solution in 500 μL was placed in individual aliquots. Water Phase: Aspirin 1–3 mg dissolved in PVA 1% or Prednisolone 0.02-1 mg in 10% ethanol PVA1%. It was DNA and nanoparticle well transfection noted that adding additional solvents to increase drug On the day before transfection, cells were placed in 12 load in this phase resulted in failure to maintain particle well plates with each well seeded at a density of 500,000 integrity and stability. myocytes in 1 mL of DMEM (GIBCO) media without an- Oil Phase: The process was very flexible in terms of tibiotics. The transfection complexes were then prepared: changing the amount of polymer added to the system Complex 1 – One 6 μg of DNA aliquot was diluted and was stable in the range of 20 – 120 mg of either into 100 μL of media in an individual eppendorf vial. PLGA type. Fargnoli et al. Journal of Translational Medicine 2014, 12:171 Page 5 of 9 http://www.translational-medicine.com/content/12/1/171 Outer Water Phase: The PVA in the system acts as a narrow, of high quality and was as follows: PLGA50:50 vital stabilizer that can be readily increased. Increases Prednisolone [234 ± 9 nm], PLGA65:35 Prednisolone beyond 2% tended to inhibit the amount but not the [228 ± 7 nm], PLGA50:50 Aspirin [323 ± 13 nm] and quality of generated nanoparticles. Thus, a working PLGA65:35 Aspirin [302 ± 7 nm]. ANOVA indicated range of 0.5-2% of PVA stabilizer in the outer water significance between the groups, specifically it was phase is suitable for accommodating various drug/polymer determined that aspirin contributed to larger particles complexes with good stability. as both PLGA50:50 and PLGA 65:35 types were signifi- cantly larger than their matched prednisolone counter- Particle characterization parts. (Figure 2C) This difference in size was most The results presented here summarize the characterization likely attributable to both higher aspirin mass content for each of the 4 resultant nanoparticle types acquired from and charge of the first water phase in the reaction 5 consecutive runs. Polymer load was fixed at 60 mg, sincesize was unaffected by theadditionof morepoly- aspirin 3 mg and prednisolone at 1 mg respectively. mer (data not shown). SEM images from the various runs for each nanoparticle Yields were very consistent and proportional to polymer type were loaded into ImageJ software for analysis. The mass input in the range of 75-80% recovery upon final har- process consistently yields uniform, spherically shaped vest. The average yields per polymer/drug type based on formulations (Figure 2A,B). The size distribution was very 60 mg input were: PLGA50:50 Prednisolone [46 ± 1 mg], Figure 2 SEM Characterization Sizing Results. A. Narrow size distribution and high quality spherical shaped yield example in A at 2μm scaling. B. Close up 500 nm scaling image indicates narrow size distribution in the 200-350 nm range consistently for all manufactured yields. C. Nanoparticle average size by drug and polymer combination. Aspirin nanoparticles of either 50:50 or 65:35 type had greater size (p<0.05) versus prednisolone. Fargnoli et al. Journal of Translational Medicine 2014, 12:171 Page 6 of 9 http://www.translational-medicine.com/content/12/1/171 PLGA65:35 Prednisolone [45 ± 2 mg], PLGA50:50 Aspirin [48 ± 1 mg] and PLGA65:35 Aspirin [47 ± 2 mg]. Produc- tion yields with increased or decreased polymer loading revealed the same results (data not shown). Loading Efficiency results were uniform for all 4 nano- particle types, independent of drug or polymer and were: PLGA50:50 Prednisolone [88.9 ± 0.01%], PLGA65:35 Pred- nisolone [88.2 ± 0.01%], PLGA50:50 Aspirin [89.0 ± 0.01%] and PLGA65:35 Aspirin [88.8 ± 0.01%]. Stability analysis Figure 4 Controlled release study results demonstrate that Positive nanoparticle visualization was realized on the aspirin particles overall release faster than prednisolone types. SEM 24 hours after re-constituting freeze dried product in saline for all 4 polymer configurations. The particle shape and size was retained. limited by a number of key variables. Therefore, produc- The stability of the nanoparticles in suspension was tion with major deviations with the water phase I input moderate to good in the range of −30 to −53 mV. A (data not shown) resulted in lower quality profiles featur- score much less than −30 indicates a stability issue with ing aggregation and wider size ranges. The first major crit- a pharmaceutical dispersion, while any score higher ical variable was the concentration of the loading drug in than −60 indicates maximum. The potential scores by the first water phase, which was largely limited by the nanoparticle type shown in Figure 3 were as follows: inherent solubility at room temperature. In the case of as- PLGA50:50 Prednisolone [−47 ± 5 mV], PLGA65:35 pirin, without solvents added, the maximum concentration Prednisolone [−31 ± 1 mV], PLGA50:50 Aspirin [−45 ± was 3 mg/mL directly at the solubility limit. Runs at the 0.5 mV] and PLGA65:35 Aspirin [−32 ± 0.9 mV]. Stat- 5–10 mg/mL range resulted in aggregation and lower istical tests revealed that the PLGA50:50, independent quality. In the case of prednisolone, it was anticipated of drug load was superior compared with the 65:35 that on a per gram basis at least 1 mg/mL would be re- type. quired to achieve high quality in addition to a realistic dosing paradigm for a rodent heart with target of 1 Controlled release of the nanoparticle formulations gram mass. This was achieved suitably with 10% Etha- Figure 4 shows a graphical depiction of the release over nol, however concentrations greater than 25% in an the span of 5 days. It was evident that the aspirin attempt to load more drug distorted the process (data released faster overall as compared with prednisolone. not shown). The PLGA and PVA stabilizer system as This is most likely due to a combination of factors presented here is therefore open to excipient manipu- including size, stability and charge. The PLGA50:50 lation provided that solubility and other attributes of Aspirin type had the fastest release profile. the selected drug are addressed such to prevent devia- tions in overall quality which may or may not be de- Process limitations sired depending on the application. We anticipate this The high quality in terms of particle shape uniformity, platform would be open for further experimentation by yield, surface charge and release properties were critically professional formulation scientists tailored to each specific PLGA/drug selection for the intended direct injection application. In-vitro myocyte transfection All wells were checked for viability and it was deter- mined that none indicated any major media discolor- ation or visual evidence of contamination. The following 5 groups all had positive detection of GFP (green) in at least 2/3 replicate wells at both 24 and 48 hours, with a greater degree of cells positive as expected at 48 hours. Figure 5 depicts independent uptake of both GFP plas- mid and nanoparticle co-signal. The absorption clusters Figure 3 Nanoparticle zeta potential colloidal stability testing were confirmed in the center of myocytes. The multiple results indicate that the PLGA50:50 nanoparticles are more DNA and nanoparticle infection groups yielded nano- stable in solution versus the PLGA65:35 types. particle presence (red) or both (yellow) at the 24 and 48 Fargnoli et al. Journal of Translational Medicine 2014, 12:171 Page 7 of 9 http://www.translational-medicine.com/content/12/1/171 regions, which are characterized by a high degree of inflammation and fibrosis. The second key finding in the final test was that PLGA uptake and release of anti- inflammatory agents in myocytes does not interfere with the absorption and trafficking of the GFP plasmid. Muscle tissue has a high risk of developing an adaptive immune response to gene products. Wilson et al. described in detail the host response after AAV delivery by route of administration and more specifically the role of inflam- mation [27]. A key finding with AAV mediated gene transfer was that the host either induces tolerance or an adaptive immune reaction through a series of com- plex interactions [28-30]. A prime risk factor in these interactions that was found to trigger adaptive immune responses were inflammatory cytokines and signals ei- ther already present in tissue or induced at the time of delivery [31]. It has been postulated that with attenu- ation of innate inflammatory response signals, the im- mune system has a much lower risk for mounting maladaptive T cell responses. Using the example of Figure 5 In Vitro Fluorescent Imaging at 48 hours post AAV, once vector capsid antigens are cleared from the transfection. All 4 particle systems exhibited safe and robust uptake in myocytes while not interfering with plasmid uptake and system, typically 12–16 weeks after delivery, there is a subsequent GFP expression. Yellow signal indicates co-existence of good chance for therapeutic tolerance. The risk is that GFP and nanoparticle in: A. PLGA65:35 Aspirin B. PLGA50:50 Aspirin an adaptive immune response will destroy those cells C. PLGA50:50 Prednisolone D. PLGA65:35 Prednisolone. expressing the transgene of interest well before these antigens are cleared. Use of anti-inflammatory agents to mitigate theinnateresponsetoinjuryislikelytore- post transfection: I. Control GFP DNA only (data not sult in enhanced long term gene expression. Intraven- shown) II. GFP and PLGA65:35 Aspirin (Figure 5A) ous delivery approaches are associated with a lower III. GFP and PLGA50:50 Aspirin (Figure 5B) IV. GFP level of induced inflammation but are also very ineffi- and PLGA50:50 Prednisolone (Figure 5C) V. GFP and cient. In contrast, the IM route in the heart remains at- PLGA65:35 Prednisolone (Figure 5D). tractive because greater cardiac specificity can be achieved, especially for angiogenesis or regenerative therapies that Discussion require a more local delivery profile. Yet IM delivery is This study presents two key findings that have broad im- associated with a more robust innate immune response plications for the advancement of cardiac gene therapies. due to associated tissue injury. First a reproducible, simple to use lab scale process was Direct injection into healthy or ischemic myocardial developed to generate anti-inflammatory nanoparticles regions introduces the gene product into a highly in- of very high quality for co-administration with gene flammatory region, which likely explains the poor car- products in a - regulatory friendly - PLGA platform. Al- diac gene therapy results with IM interventions. Early though only two anti-inflammatory drugs were utilized studies by Snyder et al. [32] reported that very little suc- in this feasibility assessment, it is anticipated that any cessful transfer occurs in damaged muscle in the inflam- matory environment. Numerous examples have validated other drug indicated for injection into muscle could be introduced by modification of the first drug water phase. these observations in gene therapy trials. In hemophilia Also the process offers an easy means to adjust the poly- trials for example it was found that IM injection into skeletal muscle resulted in transient therapeutic gene mer content in the oil phase for the desired degrad- ation/release profile, along with increasing the amount expression and an adaptive CD4+ immune response of stabilizer. Therefore this system can provide a plat- [33,34]. However, delivery of the same product infused into the liver has resulted in better outcomes and lim- form to guide future pre-clinical studies to investigate reliable clinical interventions to address the role of in- ited reactions. Muscular dystrophy trials have encoun- flammation on the relative performance of gene products tered similar difficulties and have attempted to utilize immunosuppressant drugs and other agents to limit in muscle tissue. The potential role of inflammation should not be overlooked, particularly in myocardial tissue responses after multiple IM injections compromising where the most common delivery scenario is in ischemic patient safety [35]. Fargnoli et al. Journal of Translational Medicine 2014, 12:171 Page 8 of 9 http://www.translational-medicine.com/content/12/1/171 Conclusions References 1. Gheorghiade M, Bonow RO: Chronic heart failure in the United States: a In this proof of concept study, GFP plasmid was utilized manifestation of coronary artery disease. Circulation 1998, 97:282–289. to simulate a therapeutic construct understanding that 2. Frazier OH, Myers TJ: Surgical therapy for severe heart failure. Curr Probl Cardiol naked DNA is likely to be at the lower end in terms of 1998, 23:721–768. 3. Delgado HD, Rao V, Ross JR, Verma S, Smedira N: Mechanical circulatory transduction efficiency. For more practical gene therapy assistance (State of Art). Circulation 2002, 106:2046–2050. applications, it is anticipated that viral vectors encoding 4. Siegenthaler PM, Martin J, Loo A, Doenst T, Bothe W, Beyersdorf F: the gene of interest could readily be combined with par- Implantation of the permanent jarvik-2000 left ventricular assist device. JACC 2002, 39:1764–1772. ticles containing potent anti-inflammatory drugs. The 5. Rose EA, Gelijns AC, Moskowitz AJ, Heitjan DF, Stevenson LW, Dembitsky W, hypothesis offered is that with the right formulation, the Long JW, Ascheim DD, Tierney AR, Levitan RG, Levitan RG, Watson JT, anti-inflammatory agent would be released at the suffi- Ronan NS, Meier P: Long-term use of a left ventricular assist device for end-stage heart failure. N Engl J Med 2001, 345:1435–1443. cient level over the critical post-delivery inflammatory 6. Schmia MC, Gumpert AM, Kock WJ: Cardiovascular gene therapy for period to provide an optimal viral vector trafficking myocardial infarction. Expert Opin Biol Ther 2014, 14:183–195. microenvironment. There would be a predicted increase 7. Kawase Y, Ly HQ, Prunier F, Lebeche D, Shi Y, Jin H, Hadri L, Yoneyama R, Hoshino K, Takewa Y, Sakata S, Peluso R, Zsebo K, Gwathmey JK, Tardif JC, in transduction efficiency, minimize the innate and adap- Tanguay JF, Hajjar RJ: Reversal of cardiac dysfunction after long-term tive immune response to the vector and/or transgene and expression of SERCA2a by gene transfer in a pre-clinical model of promote long term gene expression in the target tissues. heart failure. J Am Coll Cardiol 2008, 51(11):1112–1119. 8. Rengo G, Lymperopoulous A, Zincarelli C, Donniacuo M, Soltys S, This strategy of course would not be without its own limi- Rabinowitz JE, Koch WJ: Myocardial adeno-associated serotype 6 BARKct tations and would require much more experimentation gene therapy improves cardiac function and normalizes the neurhormonal to determine the best matched drug and release profile axis in chronic heart failure. Circulation 2009, 119(1):89–98. 9. Giacca M, Zacchigna S: VEGF gene therapy:therapeutic angiogenesis in for co-administration into the heart. More complex the clinic and beyond. Gene Ther 2012, 19(6):622–629. approaches in managing the host response following 10. Eulalio A, Mano M, Dal Ferro M, Zentilin L, Sinagra G, Zacchigna S, Giacca M: therapy have been applied, however it may turn out Functional screening identifies miRNAs inducing cardiac regeneration. Nature 2012, 492(7249):376–381. that simply addressing the innate immune response at 11. Wang W, Li W, Ma N, Steinhoff G: Non-viral gene delivery methods. the time of delivery may be a meritorious approach to Curr Pharm Biotechnology 2013, 14(1):46–60. advance successful clinical translation. 12. Su CH, Wu YJ, Wang HH, Yeh HI: Non-viral gene therapy targeting the cardiovascular system. Am J Physiol Heart Circ 2012, 303(6):629–638. 13. Katz MG, Swain JD, Tomasulo CE, Sumaroka M, Fargnoli A, Bridges CR: Competing interests Current Strategies for Myocardial Gene Delivery. J Mol Cell Cardiol 2011, The authors declare that they have no competing interests. 50(5):766–776. Epub 2010 Sep 15. 14. Katz MG, Fargnoli AS, Williams RD, Bridges CR: The road ahead: working towards effective clinical translation of myocardial gene therapies. Authors’ contributions Ther Deliv 2014, 5(1):39–51. doi: 10.4155/tde.13.134. ASF: Process development, particle evaluation, executed in vitro testing, 15. Logeart D, Hatem SN, Heimburger M, Le Roux A, Michel JB, Mercadier JJ: wrote manuscript. AM: Performed myocyte harvest and quality control; study How to optimize in vivo gene transfer to cardiac myocytes: mechanical design input. MGK: Study design input, manuscript drafting and revisions. or pharmacological procedures? Hum Gene Ther 2001, 12:1601–1610. RDW: Quantitative sizing analysis, figure generation, manuscript editing. KBM: 16. Katz MG, Fargnoli AS, Williams RD, Bridges CR: Gene therapy for enhancing Study design input, myocyte evaluation, manuscript editing. DBW: GPF viral and nonviral vectors for cardiac diseases: Current concepts and plasmid design and evaluation of results, manuscript editing. SY: future applications. Hum Gene Ther 2013, 24(11):914–927. Nanotechnology evaluations, experimental design, process development 17. Jessup M, Greenberg B, Mancini D, Cappola T, Pauly DF, Jaski B, Yaroshinsky A, techniques, manuscript editing. CRB: Study design and manuscript editing. Zsebo KM, Dittrich H, Hajjar RJ: Calcium Upregulation by Percutaneous All authors read and approved the final manuscript. Administration of Gene Therapy in Cardiac Disease (CUPID): a phase 2 trial of intracoronary gene therapy of sarcoplasmic reticulum Ca2+-ATPase in patients with advanced heart failure. Circulation 2014, 124(3):304–313. Acknowledgements doi: 10.1161/CIRCULATIONAHA.111.022889. Epub 2011 Jun 27. The preparation of this article was supported by the NIH grant 18. Byrne MJ, Power JM, Preovolos A, Mariani JA, Hajjar RJ, Kaye DM: 2R01HL083078-05, and partially by the James H. Heinemann Foundation. The Recirculating cardiac delivery of AAV2/1SERCA2a improves myocardial authors acknowledge the National Heart, Lung, and Blood Institute Gene function in an experimental model of heart failure in large animals. Therapy Resource Program. Marina Sumaroka PhD and Vladimir Shuvaev PhD Gene Ther 2008, 15(23):1550–1557. at the University of Pennsylvania provided assistance and consultation with 19. Kaye DM, Preovolos A, Marshall T, Byrne M, Hoshijima M, Hajjar R, Mariani JA, the microscopy evaluations. Jamie Ford PhD in the Singh Nanotechnology Pepe S, Chien KR, Power JM: Percutaneous cardiac recirculation-mediated Center at the University of Pennsylvania, provided guidance with the gene transfer of an inhibitory phospholamban peptide reverses advanced scanning electron microscopy applications. heart failure in large animals. J Am Coll Cardiol 2007, 50(3):253–260. 20. Bridges CR: 'Recirculating cardiac delivery' method of gene delivery Author details should be called 'non-recirculating' method. Gene Ther 2009, 16(7):939–940. Thoracic and Cardiovascular Surgery, Sanger Heart & Vascular Institute, Epub 2009 Apr 2. Carolinas Healthcare System, Charlotte, NC, USA. Internal Medicine, Perlman 21. Grossman PM, Han Z, Palasis M, Barry JJ, Lederman RJ: Incomplete retention School of Medicine, University of Pennsylvania Medical Center, Philadelphia, after direct myocardial injection. Cardiovasc Intervent 2002, 55(3):392–397. PA, USA. Pathology and Laboratory Medicine, Perlman School of Medicine, 22. Mingozzi F, Maus MV, Hui DJ, Sabatino DE, Murphy SL, Rasko JE, Ragni MV, University of Pennsylvania Medical Center, Philadelphia, PA, USA. Materials Manno CS, Sommer J, Jiang H, Pierce GF, Ertl HC, High KA: CD8(+) T-cell Science Engineering, School of Engineering & Applied Science, University of responses to adeno-associated virus capsid in humans. Nat Med 2007, Pennsylvania, Philadelphia, PA, USA. 13:419–422. Received: 31 March 2014 Accepted: 10 June 2014 23. Gao G, Wang Q, Calcedo R, Mays L, Bell P, Wang L, Vandenberghe LH, Published: 16 June 2014 Grant R, Sanmiguel J, Furth EE, Wilson JM: Adeno-associated virus-mediated Fargnoli et al. Journal of Translational Medicine 2014, 12:171 Page 9 of 9 http://www.translational-medicine.com/content/12/1/171 gene transfer to nonhuman primate liver can elicit destructive transgene-specific T cell responses. Hum Gene Ther 2009, 20(9):930–942. doi: 10.1089/hum.2009.060. 24. Yockman JW, Kastenmeier A, Erickson HM, Brumbach JG, Whitten MG, Albanil A, Li DY, Kim SW, Bull DA: Novel polymer carriers and gene constructs for treatment of myocardial ischemia and infarction. J Control Release 2008, 132(3):260–266. 25. Luo K, He B, Wu Y, Shen Y, Gu Z: Functional and biodegradable dendritic macromolecules with controlled architectures as nontoxic and efficient nanoscale gene vectors. Biotechnol Adv 2014, 32(4):818–830. doi: 10.1016/j. biotechadv.2013.12.008. Epub 2014 Jan 3. 26. Makadia HK, Siegel SJ: Poly Lactic-co-Glycolic Acid (PLGA) as Biodegradable Controlled Drug Delivery Carrier. Polymers (Basel) 2011, 3(3):1377–1397. 27. Mays LE, Wilson JM: The complex and evolving story of T cell activation to AAV vector-encoded transgene products. Mol Ther 2011, 19(1):16–27. Epub 2010 Nov 30. 28. Jooss K, Yang Y, Fisher KJ, Wilson JM: Transduction of dendritic cells byDNA viral vectors directs the immune response to transgene products in muscle fibers. J Virol 1998, 72:4212–4223. 29. Zhang Y, Chirmule N, Gao G, Wilson J: CD40 ligand-dependent activation of cytotoxic T lymphocytes by adeno-associated virus vectors in vivo: role of immature dendritic cells. J Virol 2000, 74:8003–8010. 30. Wang L, Dobrzynski E, Schlachterman A, Cao O, Herzog RW: Systemicprotein delivery by muscle-gene transfer is limited by a local immune response. Blood 2005, 105:4226–4234. 31. Yuasa K, Yoshimura M, Urasawa N, Ohshima S, Howell JM, Nakamura A, Hijikata T, Miyagoe-Suzuki Y, Takeda S: Injection of a recombinant AAV serotype 2 into canine skeletal muscles evokes strong immune responses against transgene products. Gene Ther 2007, 14(17):1249–1260. Epub 2007 Jun 21. 32. Snyder RO, Spratt SK, Lagarde C, Bohl D, Kaspar B, Sloan B, Cohen LK, Danos O: Efficient and stable adeno-associated virus-mediated transduction in the skeletal muscle of adult immunocompetent mice. Hum Gene Ther 1997, 8:1891–1900. 33. Wang L, Cao O, Swalm B, Dobrzynski E, Mingozzi F, Herzog RW: Major role of local immune responses in antibody formation to factor IX in AAV gene transfer. Gene Ther 2005, 12:1453–1464. 34. Cao O, Hoffman BE, Moghimi B, Nayak S, Cooper M, Zhou S, Ertl HC, High KA, Herzog RW: Impact of the underlying mutation and the route of vector administration on immune responses to factor IX in gene therapy for hemophilia B. Mol Ther 2009, 17:1733–1742. 35. Wang Z, Kuhr CS, Allen JM, Blankinship M, Gregorevic P, Chamberlain JS, Tapscott SJ, Storb R: Sustained AAV-mediated dystrophin expression in a canine model of Duchenne muscular dystrophy with a brief course of immunosuppression. Mol Ther 2007, 15(6):1160–1166. Epub 2007 Apr 10. doi:10.1186/1479-5876-12-171 Cite this article as: Fargnoli et al.: Anti-inflammatory loaded poly-lactic glycolic acid nanoparticle formulations to enhance myocardial gene transfer: an in-vitro assessment of a drug/gene combination therapeutic approach for direct injection. Journal of Translational Medicine 2014 12:171. Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit

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Journal of Translational Medicine – Springer Journals

Published: Jun 16, 2014

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