A spatio-temporal cardiomyocyte targeted vector system for efficientdelivery of therapeutic payloads to regress cardiac hypertrophyabating bystander effect

Santanu Rana a, Kaberi Datta a, Teegala Lakshminarayan Reddy b, Emeli Chatterjee a, Preeta Sen a,Manika Pal-Bhadra b, Utpal Bhadra c, Arindam Pramanik d, Panchanan Pramanik d,Mamta Chawla-Sarkar e, Sagartirtha Sarkar a,⁎a Department of Zoology, University of Calcutta, 35, B.C. Road, Kolkata 700019, Indiab Centre for Chemical Biology, Indian Institute of Chemical Technology, Uppal Road, Hyderabad 500007, Indiac Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad 500007, Indiad Department of Chemistry, Indian Institute of Technology, Kharagpur 721302, Indiae Division of Virology, National Institute of Cholera and Enteric Diseases, P-33, C.I.T. Road Scheme-XM, Beliaghata, Kolkata 700010, India

a b s t r a c ta r t i c l e i n f o

Article history:Received 15 November 2014Received in revised form 9 December 2014Accepted 5 January 2015Available online 7 January 201

Keywords:CardiomyocyteCardiac tissue engineeringDrug deliveryGene therapyCarboxy methyl chitosan

Diverse array of therapeutic regimens, drugs or siRNA, are commonly used to regress cardiac hypertrophy,although, bystander effect and lower retention of bioactivemolecules significantly reduce their functional clinicalefficacy. Carvedilol, a widely used and effective anti-hypertrophic drug, simultaneously blocks β-adrenergicreceptors non-specifically in various organs. Likewise, non-specific genome-wide downregulation of p53 expres-sion by specific siRNA efficiently abrogates cardiac hypertrophy but results in extensive tumorigenesis affectingbystander organs. Therefore, delivery of such therapeutics had been a challenge in treating cardiovascular dys-function. Cardiac tissue engineeringwas successfully accomplished in this study, by encapsulating such bioactivemolecules with a stearic acid modified Carboxymethyl chitosan (CMC) nanopolymer conjugated to a homingpeptide for delivery to hypertrophied cardiomyocytes in vivo. The peptide precisely targeted cardiomyocyteswhile CMC served as the vector mediator to pathological myocardium. Controlled delivery of active therapeuticpayloads within cardiomyocytes resulted in effective regression of cardiac hypertrophy. Thus, this novel nano-construct as a spatio-temporal vector would be a potential tool for developing effective therapeutic strategieswithin cardiac micro-environment via targeted knockdown of causal genes.

© 2015 Elsevier B.V. All rights reserved.

1. Introduction

Cardiac hypertrophy is a maladaptive response to biomechanicalstress that normalizes wall tension and thereby sustains cardiac output[1]. Prolonged hypertrophic stimulus is associated with pathologicalprogression to heart failure and premature death. Current therapeutictreatment reliesmostly on the usage of drugs and surgical interventions.Since cardiovascular diseases have been linked to various genes, smallinterfering RNA (siRNA) as small molecule drugs has garnered increas-ing interest in gene therapy off late. Earlier studies validated the thera-peutic potential of siRNAs in mice using hydrodynamic intravenousinjections for gene knockdown [2]. During conventional administrationtherapeutic molecules localize different organs of the body, though clin-ical effectiveness depends on the action of drugs on diseased tissues atdesired concentration. Selecting receptor specific drug or tissue specific

siRNA is beneficial but its effectiveness increases many folds via tissuetargeted delivery of nano-polymer encapsulated therapeutics [3].Likewise, myocardium specific controlled spatio-temporal delivery ofdrug/siRNA in vivo would result in increased concentration of drug tothe target tissue without any bystander activity, improving clinical ef-fectiveness of therapeutics.

Non-viral vectors viz. nano-polymers are non-immunogenic andconfer typical advantages such as, small size, extended circulation and ef-ficient delivery [4]. Unfortunately, in the past years very few attemptshave been made towards targeted therapy of cardiac pathology by suchvectors. It may be due to the non-availability of such cardiac tissuetargeted ligands. The present study reports development of ‘magic bullets’[5] by conjugating nanopolymers with a cardiomyocyte targeted homingpeptide for in situ delivery of therapeutics within myocardium.

Homing peptides, isolated by phage display technology are reportedfor high affinity towards specific targets in different cell types [6]. In thepresent study, a 20-mer peptide (WLSEAGPVVTVRALRGTGSW) withspecificity towards cardiac tissue [7] was conjugated with stearicacid modified CMC, a nano-polymer, to encapsulate therapeutics and

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⁎ Corresponding author at: Department of Zoology, University of Calcutta, 35,Ballygunge Circular Road, Kolkata 700019, West Bengal, India.

E-mail address: sagartirtha.sarkar@gmail.com (S. Sarkar).

http://dx.doi.org/10.1016/j.jconrel.2015.01.0080168-3659/© 2015 Elsevier B.V. All rights reserved.

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168 S. Rana et al. / Journal of Controlled Release 200 (2015) 167–178

targeted delivery within diseased myocardium for regression of cardiachypertrophy. CMC was chemically modified from chitosan; a naturallyoccurring soluble cationic polysaccharide widely used in drug deliverysystems [8] and is biocompatible, non-inflammatory, non-toxic andbiodegradable [9]. Chitosan and its derivatives encapsulate drugs [10]and entrap nucleic acids by strong electrostatic interaction with posi-tively charged amines [11]; and its protonated amine groups transportcargoes across cell membrane [12]. The nano-construct with cardiac tis-sue selective drug action enhances functional efficacy of therapeuticsensuring safer drug delivery.

Carvedilol, a non-selective β-blocker [13] with potent anti-hypertrophic action has prominent side effects to other organs [14,15].On the other hand, p53 has been reported to be a potential candidatefor cardiac dysfunction. Also, p53 null mice showed successful regressionof cardiac hypertrophy [16] but led to ectodermal tumorigenesis in suchanimals [17]. The bioactivemacromolecular drug and p53 siRNAwere se-lected as test molecules in our study encapsulated by the nano-constructfor cardiac selective delivery and efficient regression of pathological hy-pertrophy without bystander effect.

2. Methods

Detailed protocols are provided in the Supplementary material andmethods online.

2.1. Animals used

24 week old Wistar rats (Rattus norvegicus) used in this study wereprocured from NIN, Hyderabad, India. The investigation follows Guide-lines for the Care and Use of Laboratory Animals published by the USNational Institute of Health (NIH Publication no. 85-23, revised 1996)andwas also approved by the Institutional Animal Ethics Committee, Uni-versity of Calcutta (Registration no. 885/ac/05/CPCSEA), registered underCPCSEA, MoEF, GOI.

2.2. Preparation and characterization of the targeted delivery system

Stearic acid modified CMC [18] was prepared from low molecularweight Chitosan (Sigma-Aldrich), where di-tert-butyl dicarbonate(BOC2O) protected unreacted amine groups. Peptides were added tothe amine protected stearic acid modified CMC solution in the presenceof EDC and NHS. Carvedilol (Sigma-Aldrich) was loaded onto CMC ata weight ratio of 1:10 and dialyzed for 3 h, and p53 siRNA (Cat#SI02047290, Qiagen) was added to CMC-peptide at a weight ratio of1:100 and incubated for 2 h at 4 °C. These nano-constructs were charac-terized by size exclusion chromatography (SEC) (Shimadzu), dynamiclight scattering (DLS; DynaPro NanoStar™, Wyatt Technology), scan-ning electronmicroscopy (Hitachi VP-SEM S-3400N) and zeta potential(Möbiuζ™ Mobility Instrument, Wyatt Technology).

Drug loading efficiency (LE%) of Carvedilol/CMC-peptide was cal-culated by dividing Carvedilol released from CMC-peptide by totalCarvedilol loaded initially [19]. The concentration of siRNA was cal-culated by dividing siRNA entrapped within CMC-peptide by siRNAadded for conjugation [20]. Further, the release efficiency of thera-peutics was studied under the influence of pH and the stability ofnano-constructs was calculated by serum resistance and polyaniondecomplexation assay.

2.3. Isolation of neonatal cardiomyocyte (NCM)

Neonatal cardiomyocytes (NCM) isolated from 2–3 day old rat pupswere cultured on laminin (Sigma-Aldrich) coated culture plates. On daythree, NCMs were serum starved with DMEM for 12 h, before experi-mentation [21].

2.4. Peptide localization and internalization assay onto NCM

The localization efficiency of FITC tagged peptides to NCM (n = 5)was examined by incubating with 50 μmol of 20-mer peptide, scram-bled peptide and FITC for 6 h.

For internalization of CMC-peptide within cells, NCMs (n= 5) weretreated with CMC-FITC and CMC-peptide-FITC for 6 h. NCMs were pre-incubated in another plate by peptideswithout FITC for 2 h before treat-ment with CMC-peptide-FITC. After treatment, NCMs were fixed with4% paraformaldehyde and counter-stained with α-sarcomeric actinin(α-SA) before mounting with DAPI (Vector Laboratories) for imagingunder fluorescence microscope (Olympus BX51, Progres® C5) [22].

Lysosomal internalization study with NCMs (n = 5) were done byincubating CMC-peptide-FITC with 500 nM of LysoTracker® RedDND-99 (Molecular Probes) at 4 °C and 37 °C for 2 h. Identical illuminationsettings including exposure time (10 s for cell) were used.

2.5. Bio-panning of nano-constructs in vivo

Rats (n = 10) were injected with CMC-FITC, CMC-scrambledpeptide-FITC and CMC-peptide-FITC at a dose of 2 mg/kg Body Weight(BW) via tail vein and were sacrificed after periodic intervals to collectserum and tissues from organs viz. heart, liver, kidney, brain andlungs. Cryosections (6 μm) were prepared after fixation by 1:1 ace-tone/methanol and cryopreservation was done by sucrose gradientunder dark for different tissues [23]. Slides with tissue sections wereused for microscopic analysis, whereas, FITC concentration from tissuelysates and serum samples was assayed by fluorimetry.

2.6. Generation of cardiac hypertrophy in vitro and in vivo

Hypertrophy induction on serum starved NCMs (n = 5) were doneby incubating with 10!8 mol/L [Sar1]AngII (Bachem) for 24 h [21]. Invivo cardiac hypertrophy was generated by ligating right renal arteryof 24 week old rats (n = 10) [24]. Sham operated control rat groups(n= 10) underwent the similar surgical procedure without aortic liga-tion. Animals were kept under optimum conditions for 14 days andwere sacrificed on the 15th day after surgery.

2.7. Treatment with therapeutics in vitro and in vivo

Serum starved hypertrophied NCMs were treated with 50 μM ofCarvedilol alone and by CMC/CMC-peptide encapsulated Carvedilol. Ina separate set of experiment, hypertrophied NCMs were transfectedwith 500 pmol of p53 siRNA by Lipofectamine™ 2000 (Invitrogen)after 8 h of AngII treatment, as well as by CMC and CMC-peptide encap-sulated p53 siRNA. Transfection with non-specific siRNA (NS siRNA;Allstars Negative Control Qiagen, Cat#1027280; n = 5) was used asnegative control. Treated cells were harvested at the end of 24 h treat-ment period for immunofluorescence, Reactive Oxygen Species (ROS)generation, caspase-3 activity assay and reverse transcription polymer-ase chain reaction (RT-PCR).

Fig. 1. Cardiomyocyte specificity of peptide in vitro: (a) Immunofluorescence study showing preferential localization of FITC labelled peptide and CMC-peptide in NCMs. Binding of pre-incubatedunlabelled 20-mer peptide interferedwith CMC-peptide-FITC, confirming cardiomyocyte selectivity of peptide (n=5, each group). Cardiomyocyte specificitywas confirmedbycounter-staining the cells with alpha-sarcomeric actinin antibody (Scale Bar = 20 μm, magnification= 40!). (b) Graph showing significantly higher release of Carvedilol and p53 siRNAfrom CMC-peptide encapsulation at pH = 4.8 compared to pH = 7.2 (*p b 0.05). Carvedilol release efficiency was significantly slower within serum from encapsulated CMC-peptide(pH = 4.8) compared to Carvedilol pellets (pH = 7.2) (#p b 0.05). (c) Agarose gel showing higher serum resistance of p53 siRNA/CMC-peptide at different time points (p53 siRNA/CMC-peptide indicated by arrowhead; Panel I) whereas, free p53 siRNA without encapsulation showed rapid degradation within 1 h (Panel II). (d) Agarose gel showing higher retentionof p53 siRNA within encapsulated CMC-peptide with increasing concentrations of heparin incubation.

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For in vivo experiments, Carvedilol and p53 siRNAwith CMC or CMC-peptide encapsulation were intravenously injected at a dose of 2 mg/kgBW in renal artery ligated rats (n = 10, each group) on alternate days,starting from the 8th day till the 14th day of ligation. The heart, kidney,brain, liver and lungs were collected for further experimentation. Ratstreated with NS siRNA tagged with CMC-peptide and p53 siRNA/CMC-scrambled peptide were used as negative control and internal control,respectively (n = 10, each group). Heart weight/Body weight (HW/BW) ratio, in mg/g was calculated from heart tissue samples of differentrat groups [25].

2.8. Reverse-transcriptase PCR (RT-PCR)

Total RNA from cells/tissues was isolated using TRIzol reagent(Invitrogen) following the manufacturer's protocol. 2 μg of total RNAwas used to make cDNA using cloned AMV first strand cDNA synthesiskit (Invitrogen) and RT-PCRwas done for the target genes using specificoligonucleotide primers.

2.9. Western blotting

Proteins from ventricular tissues were extracted and separated bySDS-PAGE before being transferred to PVDF+membrane. After blockingwith 5% non-fat dry milk, membranes were incubated with primary an-tibodies in 5% BSA solution at 4 °C overnight. Afterwashing,membraneswere incubatedwith appropriate HRP-conjugated secondary antibodies(Pierce) at room temperature for 1 h. Finally, the immunoreactive bandswere visualized by enhanced chemiluminescence kit (Millipore) [26].

2.10. Caspase protease activity assay

Induced caspase-3 activity signifies higher apoptotic load withincells and has been detected by ApoAlert caspase-3 Fluorescent AssayKit (Clontech Laboratories). Control and treated samples from variousgroups (n = 10, each group) were lysed in 1X lysis buffer andcaspase-3 activity was determined as per the manufacturer's protocol.Caspase-3 inhibitor Ac-DEVD-CHO was used as an internal control forthe experiment [21].

2.11. Estimation of cellular reactive oxygen species (ROS) and calciumoverload

NCMs (n = 5, each group) were stained with DCFDA (2′,7′-dichlorofluorescin diacetate; Abcam) in 1X buffer for 45 min at 37 °Cand analysis was carried out by fluorescence microscopy and fluorime-try. For tissues, lysates from different rat groups were prepared in 1Xbuffer. ROS activity per μg of protein was measured in microplate read-er. Readingswere taken by excitation/emissionwavelengths of 485 nm/535 nm (Abcam). Tert-Butyl Hydrogen Peroxide (TBHP) was used as apositive control in ROS assay. Calcium overload was detected by Fluo-4, AM, cell permeant (Invitrogen) at an excitation of 494 nm and anemission of 506 nm. Cells were treated and then loaded with the dis-solved calcium indicator directly to incubate for 45 min at 37 °C underdark, before fluorescence microscopic analysis.

2.12. Estimation of total collagen by hydroxyproline assay

Hydroxyproline assaywas performed tomeasure total collagen con-tent from cardiac tissues of control and treatment rat groups. Hydroxy-proline content in the unknown sampleswas calculatedwith the help of

a standard curve. The amount of collagen was calculated bymultiplyinghydroxyproline content with a factor of 8.2 [24].

2.13. M-mode echocardiographic analysis

Cardiac function of lightly sedated animals from all groupswasmea-sured byM-mode analysis on a transthoracic study on the 15th day be-fore euthanization. Digitized images were obtained using an ultrasoundsystem (Vivid S5 system, GE Healthcare) for the calculation of anteriorand posterior end-diastolic wall thickness (IVSd, LVPWd), LV diastolicand systolic internal dimensions (LVDd, LVDs) and LV percent fractionalshortening (%FS). LV mass (LVM) was calculated by the standard cubeformula: LVM = 1.04[(LVDd + LVPWd + IVSd)3 ! (LVDd)3], where1.04 is the specific gravity of the muscle and %FS was calculated as[(LVDd ! LVDs) / LVDd] ! 100 [21,27].

2.14. Statistics

All the variables have been expressed as standard error of themean ± SEM. Data were analysed with one way ANOVA followed byStudent's t-test using SPSS software (14.0; IBM). Experiments were re-peated at least three times before analysis. Results with p b 0.05 wereconsidered significant.

3. Results

3.1. 20-Mer peptide as a targeting moiety to cardiomyocytes in vitro

The selective affinity of FITC labelled peptides were analysed inNCMs counter-stained with α-SA. Fluorescence analysis showed con-siderably higher peptide-FITC binding with NCMs compared to thescrambled peptide-FITC (Fig. 1a). Peptide-FITC conjugated with CMCalso showed substantial cellular uptake within cultured NCMs. Pre-incubation with unlabelled peptide before treatment with CMC-peptide-FITC showed considerably low internalization, due to pre-occupancy of peptide, compared to CMC-peptide-FITC (Fig. 1a). Further,colocalization of CMC-peptide-FITCwith LysoTracker® DND-99 indicat-ed endocytic internalization of nanoparticles within NCMs (Fig. S1).Cells incubated at 4 °C showed negligible internalization, as theendocytic machinery is blocked at 4 °C due to non-availability of ATP.Nano-construct treatment (100 μM for 24 h) showed negligible cellloss (b10 ± 5%) within cardiomyocytes as estimated by MTS assay(data not shown).

3.2. Preparation and characterization of nano-constructs

The H-form of CMC produced from chitosanwasmodified by stearicacid for hydrophobic core formation of the nanopolymer. Free aminogroups of stearic acid modified CMC were protected by BOC2O beforethe conjugation of peptides by EDC-NHS reaction, thus ensuringamidation of activated carboxylic groups of CMC by free amino groupof peptide. Trifluoroacetic acid (TFA)was used to remove BOC2O groupsfrom the nanopolymer. Carvedilol was encapsulated into the hydropho-bic core and p53 siRNA was entrapped by the high cationic nature ofCMC-peptide (Fig. S2a).

3.2.1. Biophysical characterization of nano-constructsCMC andCMC-peptide displayed similar retention periods signifying

the samples to have similar averagemolecularweights as shown by SEC(Fig. S2b). Hydrodynamic diameter, %Poly dispersity (%PD) and zeta

Fig. 2. Biopanning of CMC-peptide delivery in vivo. (a) Immunofluorescence study showing substantial localization of FITC in heart tissue compared to kidney, liver, brain and lungs fromrats injected with CMC-peptide-FITC (n = 10, each group) (scale bar = 50 μm, magnification = 40!) and haematoxylin/eosin (H/E) stained tissue section (scale bar = 100 μm,magnification=60!). Specificity of cardiomyocyteswas ascertained by counter-stainingwithα-sarcomeric actinin (inset). (b) Graph showing estimation of FITC quantified from varioustissue lysates after 6 h of incubation with conjugated nanopolymers (n = 10, each group). Localization efficiency of CMC-peptide-FITC was significantly high in heart tissues comparedother organs (*p b 0.05). (c) Graph showing a negative correlation between serum FITC concentration with FITC concentration in heart tissues of CMC-peptide-FITC treated rats (n =10) at different time points (p b 0.05, r = 0.93).

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potential were assayed for these constructs (228 nm, 15.8 and+18mVfor CMC-peptide, 235 nm, 10.2 and +28 mV for Carvedilol/CMC-peptide and 250 nm, 12.2 and +22 mV for p53 siRNA/CMC-peptide,respectively; Fig. S2c). Conjugation yield of peptide with CMC wasestimated to be 60% (SE ± 7.2) whereas, % drug loading efficiency(LE%) was 62.42% (SE± 8.4) for Carvedilol and encapsulation efficiencyof p53 siRNA was 83.5% (SE ± 9.7).

3.2.2. In vitro release of therapeutics by CMC-peptide encapsulationCarvedilol/CMC-peptide release profile in vitro at two different pH

values showed significantly higher Carvedilol release (41.64%; SE ±6.2) after 36 h at pH = 4.8 from CMC-peptide in rat serum comparedto 26.69% (SE ± 6.4) at pH = 7.2 (p b 0.05). p53 siRNA release in Na-acetate was also significantly pH responsive, showing 89.63% of siRNA(SE ± 12.1) release after 36 h at pH = 4.8, in contrast to 50.23%(SE ± 7.8) at pH = 7.2 (p b 0.05). Encapsulation of Carvedilol or p53siRNA by CMC alone showed no substantial difference in release kinet-ics, indicating no interference of peptide in therapeutics release (datanot shown). Free Carvedilol pellets on the other hand, showed 91.74%(SE ± 15.2) release in rat serum at physiological pH after 60 h as com-pared to significantly low percentage release of Carvedilol (43.99%;SE ± 8.2; p b 0.05) from Carvedilol/CMC-peptide constructs, signifyingincreased retention of therapeutics by the nanoparticles within bodyfluids (Fig. 1b).

3.2.3. Serum and polyanion resistance of p53 siRNA by CMC-peptide in vitroIncubation of p53 siRNA/CMC-peptide with rat serum for 24 h re-

vealed integrity of the CMC-peptide encapsulated siRNA on agarosegel, whereas free siRNA was degraded within 1 h of incubation(Fig. 1c). Stability of siRNA against polyanion attack was assayed by in-cubation of p53 siRNA/CMC-peptide with increasing concentrations ofheparin (0–15 IU/μg of siRNA), that revealed negligible decomplexationof siRNA fromCMC-peptide below 10 IU/μg of siRNA heparin concentra-tion (Fig. 1d).

3.3. Targeted localization of CMC-peptide in vivo

Microscopic analysis revealed substantial localization of CMC-peptide-FITC on cardiac tissue compared to other organs in vivo.Haematoxylin/eosin (H/E) staining showed no change in integrity of tis-sue architecture after CMC-peptide treatment (Fig. 2a). Fluorimetricanalysis of FITC signal from various tissue lysates of CMC-peptide-FITCinjected animals showed significantly higher signals in cardiac tissuecompared to other organs, whereas, CMC-FITC showed no tissue speci-ficity (p b 0.05, Fig. 2b).

Serum kinetics of nano-construct showed 50% reduction in FITCserum concentration within 30 min of injection indicating rapidclearance or endocytosis within the system, along with a proportionalincrease of FITC signal within cardiac tissue at similar time points(p b 0.05, r = 0.93, Fig. 2c).

3.4. Quantification of selective and effective delivery of therapeutics byCMC-peptide

Carvedilol/CMC-peptide or CMC-peptide entrapped Cy3-NS siRNAtreatment in vivo showed significantly higher free drug/Cy3 signal con-centration within cardiac tissues compared to other organs (p b 0.05;Fig. 3a). No significant difference in localization efficiency of both

Carvedilol and Cy3-NS siRNA was observed among different organs byCMC alone or tagged with scrambled peptide.

Significantly induced calcium overloading during hypertrophy wasefficiently downregulated by either Carvedilol or p53 siRNA taggedCMC-peptide treatment of cardiomyocytes (Fig. 3b). Similarly, Carvediloland p53 siRNA encapsulated nano-construct showed significant regres-sion of cellular ROS generation on NCMs (2.875 ± 0.34 and 3.53 ± 0.3fold, p b 0.05) and in cardiac tissues (1.84 ± 0.12 and 2.2 ± 0.17 fold,p b 0.05; Fig. 3b,c). Also, induced Caspase-3 activity (a marker for down-stream apoptosis signalling) in hypertrophied cardiomyocytes wasshown to be significantly downregulated after therapeutics encapsulatednano-construct treatment both in vitro (1.96± 0.21 and 2.29± 0.25 fold,p b 0.05) as well as in vivo (2.11 ± 0.23 and 2.29 ± 0.26 fold, p b 0.05;Fig. 3d), indicating efficient release of bioactive molecules within cardiacmyocardium.

3.5. Bioactivity of p53 siRNA encapsulated CMC-peptide within hypertro-phic cardiomyocytes

p53 mRNA expression was significantly lower (7.6 ± 0.82 folds) inAngII treatedNCMs transfectedwith p53 siRNA/CMC-peptide comparedto NS siRNA (p b 0.05), indicating no significant interference by the 20-mer peptide on knockdown efficiency of p53 siRNA after encapsulation(Fig. S3a). Likewise, p53 siRNA/CMC-peptide administration in vivo alsoshowed significantly high p53 protein knockdown efficiency (77 ±4.8%) in cardiac tissues compared to NS siRNA/CMC-peptide treatmentduring hypertrophy (p b 0.05)with no significant change of p53 expres-sion in other major organs. However, ligated rats treated with p53siRNA/CMC showed non-specific downregulation of p53 expression inall organs (p b 0.05). In cardiac tissues, p53 knockdown efficiency byp53 siRNA/CMC (43 ± 2.9%) was significantly lower than p53 siRNA/CMC-peptide (p b 0.05, Figs. 4a, S3b). Similar trends were observed forcaspase 3 activity assay (p b 0.05; Fig. 4b) in different organs of treatedrat groups by nano-construct treatment. CMC-scrambled peptide en-capsulation of p53 siRNA showed no substantial difference in knock-down efficiency of p53 among different tissues (data not shown).Targeted knockdown of p53 in cardiac tissues by CMC-peptide had nosignificant change in expression of VEGFR I, an oncogenic marker, invarious organs compared to control tissues, whereas, non-targetedp53 siRNA treatment by CMC showed significant increase of VEGFR I ex-pression in bystander tissues (Fig. 4c). Histological analysis of variousorgans of the treated rats showed no change in tissue architecture byp53 siRNA/CMC-peptide compared to control samples (Fig. 4d).

3.6. Regression of cardiac hypertrophy by targeted delivery of therapeuticsby nano-constructs

Treatment with Carvedilol/CMC-peptide and p53 siRNA/CMC-peptide in AngII induced NCMs showed significant downregulation ofhypertrophy marker gene expression [Atrial natriuretic factor (ANF;3.6 ± 0.27 fold and 3.96 ± 0.32 fold) and Beta myosin heavy chain (!-MHC; 3.6 ± 0.31 fold and 3.8 ± 0.37 fold)] when compared tohypertrophied cardiomyocytes (p b 0.05; Fig. S4).

Moreover, significant downregulation of HW/BW (1.6 ± 0.13 and1.5 ± 0.11 fold; p b 0.05), left ventricular cardiomyocyte mean cross-sectional area (CSA: 1.43 ± 0.16 and 1.32 ± 0.015 fold; p b 0.05;Fig. 5a) and the expression of hypertrophy marker genes (ANF: 3.8 ±0.42 and 5.3 ± 0.48 fold, !-MHC: 3.3 ± 0.34 and 3.1 ± 0.29 fold)

Fig. 3. Estimation of targeteddelivery of therapeutics loaded nano-constructs. (a) Graph showing concentration of Carvedilol (Panel I) and Cy3 taggedNS siRNA (Panel II) in different tissuelysates after rats were injected with various nanopolymers. Carvedilol and Cy3 tagged NS-siRNA delivery by CMC-peptide encapsulation showed increased concentration in heart tissuescompared to other organs (*p b 0.05) whereas, non-targeted delivery of therapeutics in vivo showed no organ selectivity (n= 10, each group). (b) Immunofluorescence study showingregression in calcium overload (scale bar= 60 μm,magnification= 100!) and cellular ROS generation (scale bar= 20 μm,magnification= 40!) with CMC-peptide encapsulated ther-apeutics treatment, signifying effective release of bioactive deliverables fromCMC-peptide encapsulationwithin cellularmicroenvironment (n=5, each group). (c) Graph showingdown-regulation of cellular ROS generation in vitro (n= 5, each group) and in vivowithin cardiac tissues (n=10, each group) by CMC-peptide encapsulated therapeutics treatment (*p b 0.05).(d) Graph showing significant downregulation of Caspase 3 activity in vitro (hypertrophied NCMs; n = 5 each group) and in vivo (cardiac tissues; n = 10 each group) by CMC-peptideencapsulated therapeutics compared to hypertrophied samples (*p b 0.05). Caspase 3 activity was normalized by expression values of control samples.

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when compared to untreated hypertrophied rat hearts. Hypertrophiedrats treated with CMC-peptide encapsulated Carvedilol and p53 siRNAalso showed marked downregulated expression of fibrosis markers(collagen 1: 4.6 ± 0.48 and 3.8 ± 0.32 fold and collagen 3: 3.9 ± 0.37and 4.1 ± 0.39 fold, respectively), collagen volume fraction (CVF:1.4±0.11 and 1.36±0.14 fold; p b 0.05; Fig. 5a) and total collagen con-tent by hydroxyproline assay (1.57 ± 0.21 and 1.77 ± 0.28 fold;p b 0.05; Fig. 5b). On the contrary, cardiac contractile regulators showedupregulated expression in hypertrophied rat hearts treated with CMC-peptide encapsulated therapeutics (Phospholamban: 5.4 ± 0.48 and6.3 ± 0.59 fold and SERCA2A protein: 1.77 ± 0.2 and 1.27 ± 0.15 fold,respectively; p b 0.05; Fig. 5c,d).

M-mode Echocardiography analysis revealed significantly improvedcardiac function in hypertrophied rats treated with targeted nano-encapsulated Carvedilol and p53 siRNA with significant decrease inLVDD (1.08 ± 0.13 and 1.15 ± 0.17 fold, respectively) and LVM(1.19 ± 0.13 and 1.27 ± 0.11 fold, respectively) along with significantincrease in %FS (1.41 ± 0.13 and 1.51 ± 0.17 fold, respectively) com-pared to hypertrophied rats (p b 0.05; Fig. 6a,b).

4. Discussion

Research on cardiac pathophysiology has harnessed and zeroeddown upon a few causal molecules that can significantly alter the pro-gression of the cardiac disease forms but therapeutic modulation ofthese molecules is not yet practiced due to their bystander effects caus-ing harm to other organs. Till date, no efficient targeted delivery systemwith therapeutic payload has been developed for treating pathologicalmyocardium. This study reports for the first time, a successful, non-toxic and efficient delivery of bioactive therapeutics to the pathologicalcardiac tissue microenvironment by a cardiomyocyte specific homingpeptide [7]. The successful designing of the novel CMC-peptide con-struct benchmarks a platform to deliver encapsulated therapeutics se-lectively to myocardium without adverse effects to bystander organs.

The targeted delivery system based on a CMC conjugated 20-merpeptide with high binding affinity for cardiomyocytes (Fig. 1a), showedsequence homology to Epidermal growth factor like calcium bindingprotein (E-value 1.1) in rat and Tenascin X (E-value 1.1) in human(Homo sapiens) by specific blast analysis. To ensure conjugation ofmod-ified CMC with targeting peptide by NHS-EDC, BOC2O was used to pro-tect free amino groups of CMC as well as to prevent self-polymerizationof activated carboxylic groups in CMC, ensuring the formation of a pureCMC-peptide conjugate as revealed by SEC (Fig. S2b).

Biophysical characterization revealed homogenous size distributionand positive charge of the synthesized nano-conjugates (Fig. S2c),which increase chances of successful cellular uptake of therapeuticswith CMC-peptide encapsulation [11,12]. In vitro peptide localizationassay confirmed specificity of the peptide for NCMs, the endocyticentry of the CMC-peptide conjugate within cardiomyocytes was alsosubstantiated by lysosomal co-localization with the peptide (Fig. S1).It is postulated that, the peptide binding to matrix components on cellsurface might behave as a cell penetrating peptide [28,29] that eventu-ally gets internalized by endocytic mechanisms. Furthermore, inter-group differential fluorescence intensity of CMC-peptide-FITC, with in-creased signal from cardiac tissue compared to other important organsviz. kidney, liver, brain and lungs suggests facilitated delivery of con-struct to cardiac tissues (Fig. 2).

Once the targeted delivery of CMC-peptide to myocytes wasconfirmed, it was important to assess efficiency of the system to

encapsulate and deliver drugs for targeted therapy. Two known drugsviz. Carvedilol and p53 siRNA were tested for effective delivery withindiseased myocardium. Carvedilol is a commonly used vasodilatorybeta-blocker [13] whereas p53 siRNA can knockdown p53 gene and re-portedly plays a role during hypertrophy [16,21,30] with considerablebystander effect. Genetic knockdown of p53 in transgenic heart failuremouse model though resulted in improved cardiac function; yet suchanimals succumbed to increased tumorigenesis all over the body [19].Cardiac tissue specific knockdown of p53 has the advantage of avoidingsuch oncogenic effects (Fig. 4c,d), as observed in p53 null mice [17]. Ourstudy showed that targeted delivery of Carvedilol or p53 siRNA to path-ological myocardium with CMC-peptide encapsulation increased theaccumulation of drug specifically within cardiac tissues (Fig. 3a), alongwith effective regression of cardiac hypertrophy.

Enhanced retention of CMC-peptide encapsulated therapeuticsunder physiological conditions signified higher circulation time for thesame. The pH sensitive release kinetics of encapsulated drug andsiRNA from CMC-peptide suggests a facilitated release of Carvediloland siRNA within acidic phagosomes (pH = 4.8) from CMC-peptidesas compared to the neutral environment of the blood (pH = 7.4;Fig. 1b), suggesting an efficient release of therapeutic deliverables tothe targeted cardiomyocytes via endocytic route [31]. Consistently, re-sistance to serum-mediated degradation of CMC-peptide entrappedsiRNA (Fig. 1c) suggests increased retention and higher circulationtime for the same in vivo [32]. Also, CMC-peptide encapsulation showedincreased resistance of p53 siRNA to heparin decomplexation (Fig. 1d),conforming efficient passage of encapsulated siRNA across polyanionicbased decomplexation of cell membranes [33]. Therapeutics encapsu-lated CMC or CMC-peptide showed efficient regression of hypertrophyin NCMs (Fig. S4), as CMC is an efficient vector in cellular delivery ofdrugs and nucleic acid molecules [10,11]. Significantly reduced calciumoverloading, ROS generation and caspase-3 activity in therapeuticencapsulated CMC-peptide treated cardiomyocytes also confirmed theefficient release of therapeutics (Fig. 3c,d). These data suggests thatCarvedilol shows such intracellular pleiotropic action without bindingto β-adrenergic receptors [34], as p53 siRNA by its transient p53 knock-down within hypertrophied NCMs.

Our study clearly showed that cardiac tissue targeted delivery oftherapeutic payloads significantly regressed the hypertrophic effectswithin diseasedmyocardium as evidenced by reduced expression of hy-pertrophy markers (Fig. 5c), HW/BW ratio and improved M-modeEchocardiography parameters like reduced LVDD, LVM and increased%FS (Fig. 6). Tissue specific delivery of therapeutics in hypertrophiedrats resulted in significantly reduced cardiomyocyte CSA and collagenvolume compared to untreated hypertrophied rats (Fig. 5a,b). Improvedcontractility of myocytes in such animals was indicated by inducedexpression of phospholamban and SERCA 2A (Fig. 5c). These provideevidence to successful targeted anti-hypertrophic action of such formu-lations with significantly reduced bio-availability of therapeutics tonon-targeted tissues.

Taken together, these results strongly suggest that the CMC encap-sulated 20-mer peptide moiety facilitates targeted delivery of drugsand nucleic acid molecules within diseased myocardium, along withcardiomyocyte selective permeation for the therapeutics loaded nano-constructs, thus minimizing adverse effects to other organs. However,the precise mechanism of cardiomyocyte selective binding and inter-nalization of the 20-mer peptide needs to be investigated further.Characterization of its bindingmoiety in silicomight identify unique car-diomyocyte selective binding domain of the peptide; to shorten the

Fig. 4. Analysis of targeted p53 knockdown in vivo. (a)Western blot analysis showing significant downregulation of p53 expression in hypertrophied rat hearts compared to other organswhen treated with p53 siRNA/CMC-peptide whereas, p53 siRNA/CMC treated rats showed downregulated p53 expression in all organs. NS siRNA showed no significant change of p53expression in all organs. GAPDHwas used as internal loading control for all experiments (n= 10, each group). (b) Graph showing no significant change of Caspase 3 activity in bystanderorgans (#p N 0.05) via CMC-peptide encapsulated therapeutics (n=10, each group). (c)Western blot analysis and graph showing oncogenicmarker protein VEGFR I expression in kidney,liver, brain and lungs after delivery of p53 siRNA by heart tissue targeted CMC-peptide and non-targeted CMC encapsulation (n = 10, each group). Non-targeted p53 knockdown inbystander organs resulted in significant upregulation of VEGFR I compared to targeted p53 knockdown (*p b 0.05). (d) Photomicrographs showing no change in tissue architecture ofdifferent organs by H/E staining after p53 siRNA/CMC-peptide treatment to rats compared to nontargeted treatment (n = 10, each group) (scale bar = 40 μm, magnification = 60!).

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Fig. 5. Regression of cardiac hypertrophy in vivo by targeted delivery of therapeutics. (a) Haematoxylin/eosin (H/E) andMasson-trichrome staining of cardiac tissue sections from differenttreatment groups (n=10, each group) showing significant decrease of cross-sectional area (CSA) and collagen volume fraction (CVF) in cardiac tissues of hypertrophied rats injectedwithCMC-peptide encapsulated therapeutics compared to hypertrophied rats (scale bar = 40 μm, magnification = 60!) (*p b 0.05). (b) Graph showing significant decrease of total collagencontent by hydroxyproline assay in CMC-peptide encapsulated Carvedilol and p53 siRNA treated rat hearts compared to hypertrophied rats (n = 5, each group; *p b 0.05). (c) RT-PCRanalysis showing significant downregulation in expression of ANF, !-MHC, Collagen 1, Collagen 3 and upregulated Pln expression in ligated rat hearts targeted with therapeutics encapsu-lated CMC-peptide (n = 10, each group). GAPDHwas used as loading control. Western blot analysis revealed significantly upregulated SERCA2A expression in therapeutics encapsulatedCMC-peptide treated hypertrophied rat hearts compared to other groups (n= 10, each group). GAPDHwas used as an internal loading control. (d) Graphs showing significant change inrelative band intensity of various gene expressions by RT-PCR (ANF: *p b 0.05, !-MHC: #p b 0.05, collagen 1: §P b 0.05, collagen 3: ¶P b 0.05 and Pln: @p b 0.05) and SERCA 2A proteinexpression (#p b 0.05) by CMC-peptide encapsulated therapeutics treatment in heart tissue samples.

176 S. Rana et al. / Journal of Controlled Release 200 (2015) 167–178

targeting peptide size formore efficient physiologicalmovementwithincellular microenvironment. Targeted therapeutic delivery to cardio-myocytes thus promises to be an efficient approach for improving cardiactherapeutic strategies and lead to the development of innovative means

for formulating novel drug regimens to treat different cardiovascular dis-eases. This novel delivery method can control concentration ratio of drugtreated to that releasedwithin diseasedmyocardium, thereby providing asolution to effective drug dosing, depending on the pathophysiological

Fig. 6.M-mode echocardiographic analysis for regression of cardiac hypertrophy by targeted delivery of therapeutics. (a) RepresentativeM-mode echocardiographic images of treated anduntreated rat groups with nanoconstructs (n = 10, each group). (b) Graph showing significant change in M-mode echocardiographic parameters LVDD and %FS in different rat groups(n = 10, each group). Targeted therapeutics delivery in hypertrophied rats revealed marked decrease in LVDD and increase %FS, signifying improved cardiac function in these animals(*p b 0.05).

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state with minimal bystander effects. Thus, it promises to provide a solu-tion to the bottleneck in clinical and experimental translation of targetedtherapy of pathological heart conditions in the future.

5. Conclusion

This report demonstrates for the first time, the successful use of amyocyte targeted 20-mer peptide conjugated to stearic acid modifiedCMC that encapsulates and successfully delivers drug or siRNA foreffective treatment of pathological myocardium, without any bystandereffects. Conventional treatmentwith Carvedilol against cardiac ailmentshas been reported tomodulatemetabolic profile of liver and glomerularfiltration rate in kidney, while, non-targeted genome-wide knockdownof p53 gene has been demonstrated to improve cardiac function consid-erably, yet leads to acute oncogenesis all over the body. This novel car-diomyocyte targeted nano-construct ensures negligible delivery of suchtherapeutics to bystander organs, thereby mitigating such adverse ef-fects with efficient regression of cardiac hypertrophy. Such a nano-construct thus promises to be a potential clinical tool for on-target ther-apeutic delivery in active form and at controlled dosage for effectivemanagement of myocardial pathophysiology.

Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.jconrel.2015.01.008.

Competing interests

No conflicts of interest to disclose.

Acknowledgments

The work was funded by extramural research grants fromDepartment of Biotechnology, Government of India (grant no. BT/PR3709/BRB/10/980/2011),Department of Science and Technology,Government of India (grant no. SR/SO/HS-100/2009) andCouncil ofScientific and Industrial Research, Government of India (grant no.37(1393)/10/EMR-II). A patent application has been filed with Govern-ment of India, Patent Application No.: 0825/KOL/2013; for this finding.

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