1. Highlights:
Co-delivery of drugs-minicircle DNA by nanoparticle-in-microsphere multipart systems
Gas-generating TPGS-PLGA microspheres present a pH-responsive drug release.
This study focus on drug-DNA combinatorial therapy to target multiple cancer hallmarks
They design the carrier a stimuli-responsive character, specifically for cancer microenvironment

2. Objectives and rationales
Few materials exhibited suitable physicochemical properties to co-delivery small molecule gene combination
Peptide-functionalized DSPE/PEG liposome: paclitazel and miRNA antagomir 10b
Triblock copolymer micelle: Dox and mcDNA
design: must display hydrophilic or hydrophobic character for drug encapsulation and positively charged groups for DNA condensation
(peptide: histidine rich-protonate into positive charge in acidic environment)
Amphiphilic (CDI:carbonyldiimidazole-)
V.M. Gaspar, C. Gonçalves, D. de Melo-Diogo, E.C. Costa, J.A. Queiroz, C. Pichon, F. Sousa, I.J. Correia, Journal of Controlled Release 189 (2014)
Q. Zhang, R. Ran, L. Zhang, Y. Liu, L. Mei, Z. Zhang, H. Gao, Q. He, Journal of Controlled Release 197 (2015)

3. Objectives and rationales
Manipulating drug or gene release in a spatially and temporally controlled at the target site:
Gas-generating pH-sensitive carrier: mediated by bicarbonate salts (NaHCO3 or NH4HCO3)
Cholesterol-mPEG2000-DSPE liposome loading NH4HCO3 and Dox: delivered to multi-drug resistant breast cancer
Develop a nanoparticle in microsphere hybrid delivery system (NIMPS):
Amino acid-modified (L-histidine and L-arginine) chitosan nanoparticle: mcDNA (3.06kbp)
Doxorubin
TPGS coating: D-α-tocopherol PEG succinate, enhance cellular uptake and particle stability
Gas triggering release has been demonstrated and showed increased intracellular drug conc.
Bacterial plasmid DNA is a common choice. (include a bacterial origin of replication and an antibiotic resistance gene, transcription unit: promotor and polyA, replicate independently from host DNA)

4. McDNA An inherent supercoiled topological isoform
The lack of any bac-ORI
The absence of any sequence coding for antibiotic resistance
The existence of a therapeutic transgene whose expression is controlled by a mammalian promoter
The existence of a specific sequence attributed to the site of recombination where the precursor parental plasmid (PP) yields the minicircle
Minimalistic backbone (exclude bac-ORI, no antibiotic resistance, controlled by mammalian promoter)
Avoid any possible horizontal gene transfer, mcDNA size is smaller (2.9kbp) and 77 fold high expression compared to 52kbp
Expert opinion on biological therapy 15(3) (2015)

5. DOX-loaded gas-generating hollow microspheres
Double emulsion solvent diffusion-evaporation method: W/O/W
+hydrophobic DOX
(convert Doxorubicin hydrochloride salt by adding TEA prior to formulation)
+CH-HR-mcDNA
The NaHCO3+DOX were initially added to PVA solution and stirred.
Then PLGA in DCM (O) were dropped into aqueous phase=>sonicated=> primary emulsification
Then PVA or TPGS as surfactant were added to primary W/O emulsion=>homogenized=> then transfer to DI water and let organic solvent evaporated.

6. mcDNA amino acid-Chitosan nanoparticles
EDC/NHS coupling: primary amine group (in chitosan glucosamine) and carboxylic acid (histidine, arginine)
Complex DNA with chitosan nanoparticle by incubation for 30 min
V. Gaspar, J. Marques, F. Sousa, R. Louro, J. Queiroz, I. Correia, Nanotechnology 24(27) (2013)

7. Formulation of gas-generating PLGA microspheres
PLGA microsphere-PVA
PLGA microsphere-TPGS
TPGS: low Polydispersity than PVA=> more homogeneous
TGPS: larger size, PEG shell
TGPS: slightly decrease of zeta-potential
In accordance with previous literature

8. Formulation of gas-generating PLGA microspheres
Determined through UV-vis spectrophotometry at λ=485nm
X-ray diffraction
X-ray: No significant difference between microPVA and TGPS
Encapsulation efficiency: >95% (previously article use liquid chromatography: crizotinib, sildenafil)

9. Formulation of gas-generating PLGA microspheres
micro-PVA-DOX
micro-TGPS-DOX
After loading drug, maintain spherical shape
After loading drug: lead to smaller particle (1877nm compared to 2362nm)
Charge changed closed to neutral (-9.7) (compared to -21), affect therapeutic efficiency: previously demonstrated that particle zeta potential in the range of neutrality (+10~-10) achieve optimal for tumor penetration.

10. Microcarriers biological characterization- in vitro biocompatibility
Evaluated by resazurin assay.
HeLa cell
Positive control: HeLa cell in ethanol
Blank micro-PVA
Like alamarBlue assay, cell permeable redox indicator, viable cells with active metabolism can reduce resazurin into the resorufin product which is pink and fluorescent, slightly more sensitive than tetrazolium reduction (MTS). “usually measure fluorescence”, can be multiplexed with other methods such as measuring caspase activity to study the mechanism of cytotoxicity.
98% of viability.
Blank micro-TGPS

11. Microcarriers biological characterization- Cellular uptake
Flow cytometry and confocal microscope
Cells: stained with WGA-Alexa Fluor594 (cell surface glycoprotein)
Micro TPGS exhibits effective cell uptake

12. Cellular uptake Micro-TGPS shows 5.2 fold higher than micro-PVA
% positive cells: 5.2 fold higher cellular uptake in comparison to micro PVA, in agreement with previous literature.
Free drug particularly accumulated in the nuclear compartment, while micro PVA shows a widespread distribution (previous page).
TPGS showed a clear accumulation of drug in nuclear compartment. (Therapeutic efficacy: Dox located in nuclear to inhibits the progression of topoisomerase. II
PLGA-PVA: energy independent, macropynocytosis internalization

13. Stimuli-responsive drug release
In acid medium: a rapid and extensive Dox release
3.88 fold higher drug release at 8hr
Negligible burst release at pH7.4. Only 13% of loaded drug is released at 30h.
Retention of the drug: reduces off-target cytotoxicity effects upon their administration
Confirm the stimuli-responsive profile.

14. Stimuli-responsive drug release
PLGA-microTPGS:
preserve morphology integrity at pH7.4.
Confirm the stimuli-responsive profile.

15. Formulation of drug-gene loaded pH-responsive PLGA Microspheres (NIMPS)
mcDNA chitosan nanoparticles: assembled via electrostatic attraction (incubation)
Characterization of mcDNA chitosan nanoparticles:
DLS: 180nm
Agarose electrophoresis: lane1-free mcDNA, lane2-CH-HR-mcDNA
Zeta-potential: positive charge
1. Chitosan np can completely condense mcDNA

16. Formulation of drug-gene loaded pH-responsive PLGA Microspheres (NIMPS)
mcDNA: FITC labelled
Dual loaded showed less negative zeta potential compared to single loaded
SEM show NIMPS maintain their spherical morphology
B=3D reconstruction

17. Formulation of drug-gene loaded pH-responsive PLGA Microspheres (NIMPS)
CH-HR nanoparticles inclusion in micro-TPGS-Dox microcarriers
Co-localization of Dox and nanocarrier (FITC-mcDNA)
Colocalization analysis was performed by using thresholded mander’s colocalization algorithm (tM: ).

18. NIMPS cellular uptake and gene expression
Flow cytometry: hybrid NIMPS demonstrated higher internalization (87.1%) in comparison to micro-TPGS-Dox (72.9%)
NIMPS are localized in the intracellular compartment and that Dox is present in cell nucleus.

19. NIMPS cellular uptake and gene expression
GFP of CH-HR-mcDNA is slightly higher than NIMPS-mcDNA.
quantified by confocal microscopy
quantified by spectrofluorimetry
Maybe attributed to that mcDNA NP must be released from microocarriers before being localized.

20. Delivery systems cytotoxic activity
MTS assay
Micro-TPGS-Dox displays 1.7 fold higher cytotoxicity than Micro-PVA-Dox
NIMPS-Dox-mcDNA causes significant higher cytotoxicity.

21. Conclusions
Inclusion of TPGS as coating for PLGA improved cellular uptake.
Minicircle DNA nanoparticles inside microspheres promote transgene expression.
Lack of the release profile and carrier stability for co-delivery formulation.
Drug-gene loaded systems present higher cytotoxic activity in cancer cells.
They did not report the effective dosage

22. References
Q. Zhang, R. Ran, L. Zhang, Y. Liu, L. Mei, Z. Zhang, H. Gao, Q. He, Simultaneous delivery of therapeutic antagomirs with paclitaxel for the management of metastatic tumors by a pH-responsive anti-microbial peptide-mediated liposomal delivery system, Journal of Controlled Release 197 (2015)
V.M. Gaspar, C. Gonçalves, D. de Melo-Diogo, E.C. Costa, J.A. Queiroz, C. Pichon, F. Sousa, I.J. Correia, Poly (2-ethyl-2-oxazoline)–PLA-g–PEI amphiphilic triblock micelles for co-delivery of minicircle DNA and chemotherapeutics, Journal of Controlled Release 189 (2014)
V. Gaspar, D.d. Melo-Diogo, E. Costa, A. Moreira, J. Queiroz, C. Pichon, I. Correia, F. Sousa, Minicircle DNA vectors for gene therapy: advances and applications, Expert opinion on biological therapy 15(3) (2015)
V. Gaspar, J. Marques, F. Sousa, R. Louro, J. Queiroz, I. Correia, Biofunctionalized nanoparticles with pH-responsive and cell penetrating blocks for gene delivery, Nanotechnology 24(27) (2013)