‘Dendrimer-Cationized-Albumin’ encrusted polymeric nanoparticle improves BBB penetration and anticancer activity of doxorubicin

https://doi.org/10.1016/j.ijpharm.2018.11.035Get rights and content

Abstract

Glioblastoma is one of the most rapaciously growing cancer within the brain with an average lifespan of 12–15 months (5-year survival <3–4%). Doxorubicin (DOX) is clinically utilized as a first line drug in the treatment of Glioblastoma, however, its restricted entry into the brain via the blood–brain barrier (BBB), limited blood-tumor barrier (BTB) permeability, hemotoxicity, short mean half-life of 1–3 hr as well as rapid body clearance results in tremendously diminished bioactivity in glioblastoma. Dendrimer-Cationized-Albumin (dCatAlb) was synthesized following the carboxyl activation technique and the synthesized biopolymer was characterized by FTIR, MALDI-TOF and zeta potential. The prepared dCatAlb was encrusted on DOX-loaded PLGA nanoparticle core to develop a novel hybrid DOX nanoformulation (dCatAlb-pDNP; particle size: 156 ± 10.85 nm; ƺ: −10.0 ± 2.1 mV surface charge). The formulated dCatAlb-pDNP showed a unique pH-dependent DOX release profile, diminished hemolytic toxicity, higher drug uptake (<0.001) and cytotoxicity in U87MG glioblastoma cells, increase levels of caspase-3 gene in U87MG cells (approximately 5.35-fold higher) inferred that anticancer activity is primarily taking place through caspase-mediated apoptosis mechanism. The developed novel DOX nanoformulation also showed superior trans-epithelial permeation transport across monolayer bEnd.3 cells as well as notable biocompatibility and stability. The dCatAlb-pDNP showed enhanced BBB permeation efficacy as confirmed permeation assay in bEnd.3 cell-based model. The long-term formulation stability of developed nanoformulations was studied by storing them at 5 ± 2 °C and 30 ± 2 °C/60 ± 5% Relative Humidity (% RH) in the stability chamber for a period of 60 days (ICHQ1A (R2)). The outcomes of this investigation evidently indicate that dCatAlb-pDNP offers superior anticancer activity of DOX in glioblastoma cells while significantly improving its BBB permeation. The developed formulation is a biocompatible, safer and commercially viable approach to delivering DOX selectively in sustained manner glioblastoma while countering its hemolytic toxic effect, which is a major ongoing issue with conventional DOX injectable available in the market today.

Introduction

Glioblastoma is an aggressive primary brain tumor classified as astrocytoma grade-IV tumor (undifferentiated aggressive high-grade tumors) and comprised of 80% malignancy of tumor (Louis et al., 2016). Nowadays available treatments for glioblastoma is surgical resection, radiotherapy, and adjuvant chemotherapy which are invasive with numerous side effects (Wick et al., 2014). A chemotherapeutic agent as Doxorubicin (DOX; Adriamycin) is an anthracycline analog first-line drug in glioblastoma therapy (Lesniak et al., 2005). It suppresses the progress of cancer cells by hindering an enzyme called topoisomerase-2. However, the available literature and oncologist’s recommendations infer that the full clinical potential of DOX has still not been attained in glioblastoma therapy due to its restricted entry into the brain via the blood-brain barrier (BBB)/blood-tumor barrier (BTB). Because of higher hemotoxicity, short mean half-life (t1/2, 1–3 hr) and rapid body clearance impede its therapeutic activity (Ichikawa et al., 2014).

BBB is a lipophilic barricade, due to close-fitting connections of astrocytes and endothelial cells and also control the transportation of active molecules which are less lipid soluble and high molecular weight (>500 Da) through the BBB. Owing to tight junction and efflux transporter, the permeability from the brain endothelial cell is limited (Deeken and Löscher, 2007, Van Tellingen et al., 2015). Thus, it necessitates the development of a modality that has the capability to transport the anticancer drug through the BBB which has been a foremost challenge in research and hence be determined by emerging a nanoformulation tactic that improves the drug bioavailability in the central nervous system (CNS) (Zhang et al., 2016).

Nanomedicine has immense ability to deal the pharmaceutical and biopharmaceutical issues associated with therapeutic drugs via enhancing their therapeutic potential and targeting (Kim et al., 2015, Osswald et al., 2016). The surface engineered nanoparticles have been shown higher brain uptake via surmounting BBB with the improved therapeutic potential of the anticancer drugs in the treatment of glioblastoma (Yang et al., 2015).

Poly-(lactic-co-glycolic acid) (PLGA) is a biocompatible and biodegradable copolymer comprised of lactic acid and glycolic acid bears excellent properties as a drug carrier. Also approved by USFDA as a generally regarded as a safe and non-toxic polymer (Masood, 2016). However, PLGA nanoparticle could deliver dopamine decrease auto-oxidation and neurological defects reversed in Parkinson’s rat (Pahuja et al., 2015). Whereas, TIMP-1 protein loaded PLGA nanoparticle could not able to deliver protein across the BBB due to the insignificant opening of tight junction between brain endothelial cells. To enhance the delivery of proteins across BBB, PLGA nanoparticle were coated with polysorbate 80 (Chaturvedi et al., 2014).

In this context, surface modification of nanoparticles has a better capability to bypass the BBB via adsorptive transcytosis and electrostatic interaction. Albumin nanoparticle has excellent potential as chemotherapeutic drug vehicle for tumor delivery due to its biocompatible ability, non-immunogenic effect, non-toxic, in-vivo stability, ease of surface modification etc. (Elsadek and Kratz, 2012). Surface modification of albumin through dendrimer massively enhance the loading of payloads. Particularly, involved PAMAM dendrimer improve loading, targeting due to electrostatic interaction and also aid in achieving controlled drug release (Luong et al., 2016). PAMAM dendrimer able to protonate the free amino group at acidic pH environment of the tumor and release payloads at tumor site only (He et al., 2015). Adsorptive transcytosis via cationic bovine serum albumin has been a method of choice to by-pass the BBB without compromising the tight junction integrity of BBB (Shi et al., 2017).

In this investigation, we report a strategy to overcome the challenges associated with DOX delivery in glioblastoma by bypassing BBB by utilizing dendrimer cationized albumin (dCatAlb) encrusted with PLGA nanoparticle (dCatAlb-pDNP). In this investigation, the dCatAlb was synthesized following carboxyl activation technique and the synthesized copolymer was encrusted on DOX-loaded PLGA nanoparticle core (dCatAlb-pDNP) and characterized for hydrodynamic particle size, surface zeta potential, pH-dependent particle size analysis, and serum stability. The effect of pH on the drug release profile, hydrodynamic particle diameter and surface charge of nanoparticle over existing PLGA conventional DOX nanoparticle version is demonstrated. A meticulous explanation of the effect of time-dependent in vitro permeation, cellular uptake, cytotoxicity, hemolytic toxicity, the effect of time and pH reliant serum stability is explored and reported. The apoptosis assay using U87MG glioblastoma cancer cell model and the enhanced BBB permeation efficacy was confirmed using bEnd.3 cell-based permeation model. The long-term formulation stability of nanoparticle was also performed at 5 ± 2 °C and 30 ± 2 °C/60 ± 5% Relative Humidity (% RH) in the stability chamber for a period of 60 days (ICHQ1A (R2)).

Section snippets

Materials

DOX was acquired as a gift sample from Yarrow Chem Ltd (Mumbai, India). PLGA (50:50), 2nd generation polyamidoamine dendrimer (2.0G PAMAM), Tween 80, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and cellulose dialysis tubing (MWCO 12–14 kDa) were purchased from Sigma Aldrich (St. Louis, USA)). Albumin Fraction V, N-hydroxy succinimide (NHS), Di-methyl-sulphoxide (DMSO), Pluronic F-68 (PF-68), FITC, Triton-X 100 were obtained from HiMedia Laboratories GmbH, Germany. Methanol, Isopropyl

Results and discussion

The non-invasive nanosized bio-systems confers the interactions with cellular bio-environment and associated biomacromolecules to circumvent the exceedingly lipophilic barrier that restricts the permeation of a wide variety of biotherapeutics across the BBB (Osswald et al., 2016). The brain cerebral endothelial cells bear negative charge due to the presence of the phospholipid bilayer membranous structure. In this line, the positive charge of nanoparticle provides greater benefits to cross BBB

Conclusion

In recent years, BBB overcoming tactics and targeting to brain tumor is going to enlarge hugely. That provides numbers of chemotherapeutical approaches as well as through nano therapy accurate targeting to specific desired part of the brain could achieve with help of surface modification, peptide conjugation, magnet-based targeting etc. Formerly, our group had successfully delivered the dendrimer comprised a variety of nanoformulations. Although the great application of dendrimer in drug

Disclosures

There is no conflict of interest and disclosures associated with the manuscript.

Acknowledgment

RKT would like to acknowledge Science and Engineering Research Board (Statutory Body Established Through an Act of Parliament: SERB Act 2008), Department of Science and Technology, Government of India for a grant (Grant #ECR/2016/001964) and N-PDF funding (PDF/2016/003329) for work on targeted cancer therapy in Dr. Tekade’s Laboaratory. RKT also acknowledge the support by the Fundamental Research Grant (FRGS/1/2015/TK05/IMU/03/1) scheme of the Ministry of Higher Education, Malaysia to support

References (92)

  • A. Jain et al.

    Surface engineered polymeric nanocarriers mediate the delivery of transferrin–methotrexate conjugates for an improved understanding of brain cancer

    Acta Biomater.

    (2015)
  • P. Kesharwani et al.

    Generation dependent cancer targeting potential of poly (propyleneimine) dendrimer

    Biomaterials

    (2014)
  • P. Kesharwani et al.

    Dendrimer generational nomenclature: the need to harmonize

    Drug Discov. Today

    (2015)
  • S.-S. Kim et al.

    Effective treatment of glioblastoma requires crossing the blood–brain barrier and targeting tumors including cancer stem cells: the promise of nanomedicine

    Biochem. Biophys. Res. Commun.

    (2015)
  • E. Leo et al.

    Doxorubicin-loaded gelatin nanoparticles stabilized by glutaraldehyde: involvement of the drug in the cross-linking process

    Int. J. Pharm.

    (1997)
  • W. Lu et al.

    Cationic albumin-conjugated pegylated nanoparticles as novel drug carrier for brain delivery

    J. Controlled Release

    (2005)
  • D. Luong et al.

    PEGylated PAMAM dendrimers: enhancing efficacy and mitigating toxicity for effective anticancer drug and gene delivery

    Acta Biomater.

    (2016)
  • Y. Malinovskaya et al.

    Delivery of doxorubicin-loaded PLGA nanoparticles into U87 human glioblastoma cells

    Int. J. Pharm.

    (2017)
  • F. Masood

    Polymeric nanoparticles for targeted drug delivery system for cancer therapy

    Mater. Sci. Eng., C

    (2016)
  • J.-C. Olivier

    Drug transport to brain with targeted nanoparticles

    NeuroRx

    (2005)
  • R. Radhakrishnan et al.

    Encapsulation of biophenolic phytochemical EGCG within lipid nanoparticles enhances its stability and cytotoxicity against cancer

    Chem. Phys. Lipids

    (2016)
  • V. Sokolova et al.

    Characterisation of exosomes derived from human cells by nanoparticle tracking analysis and scanning electron microscopy

    Colloids Surf., B

    (2011)
  • F. Sousa et al.

    Nanoparticles provide long-term stability of bevacizumab preserving its antiangiogenic activity

    Acta Biomater.

    (2018)
  • S. Thakur et al.

    Impact of pegylation on biopharmaceutical properties of dendrimers

    Polymer

    (2015)
  • Y. Thasneem et al.

    Biomimetic mucin modified PLGA nanoparticles for enhanced blood compatibility

    J. Colloid Interface Sci.

    (2013)
  • O. Van Tellingen et al.

    Overcoming the blood–brain tumor barrier for effective glioblastoma treatment

    Drug Resist. Updates

    (2015)
  • H. Wang et al.

    Low-molecular-weight protamine-modified PLGA nanoparticles for overcoming drug-resistant breast cancer

    J. Controlled Release

    (2014)
  • A.K. Yadav et al.

    Development and characterization of hyaluronic acid–anchored PLGA nanoparticulate carriers of doxorubicin

    Nanomed.: Nanotechnol. Biol. Med.

    (2007)
  • G. Yan et al.

    Stepwise targeted drug delivery to liver cancer cells for enhanced therapeutic efficacy by galactose-grafted, ultra-pH-sensitive micelles

    Acta Biomater.

    (2017)
  • A. Agarwal et al.

    Cationized albumin conjugated solid lipid nanoparticles as vectors for brain delivery of an anti-cancer drug

    Curr. Nanosci.

    (2011)
  • S. Alam et al.

    Development and evaluation of thymoquinone-encapsulated chitosan nanoparticles for nose-to-brain targeting: a pharmacoscintigraphic study

    Int. J. Nanomed.

    (2012)
  • A.A. Alshatwi

    Catechin hydrate suppresses MCF-7 proliferation through TP53/Caspase-mediated apoptosis

    J. Exp. Clin. Cancer Res.

    (2010)
  • A. Asati et al.

    Surface-charge-dependent cell localization and cytotoxicity of cerium oxide nanoparticles

    ACS Nano

    (2010)
  • M. Bertolla et al.

    Solvent-responsive molecularly imprinted nanogels for targeted protein analysis in MALDI-TOF mass spectrometry

    ACS Appl. Mater. Interfaces

    (2017)
  • E.V.R. Campos et al.

    Polymeric and solid lipid nanoparticles for sustained release of carbendazim and tebuconazole in agricultural applications

    Sci. Rep.

    (2015)
  • P. Chanphai et al.

    Characterization of folic acid-PAMAM conjugates: drug loading efficacy and dendrimer morphology

    J. Biomol. Struct. Dyn.

    (2018)
  • M. Chaturvedi et al.

    Tissue inhibitor of matrix metalloproteinases-1 loaded poly (lactic-co-glycolic acid) nanoparticles for delivery across the blood–brain barrier

    Int. J. Nanomed.

    (2014)
  • M.B. Chaudhari et al.

    Solid lipid nanoparticles of amphotericin B (AmbiOnp): in vitro and in vivo assessment towards safe and effective oral treatment module

    Drug Deliv. Trans. Res.

    (2016)
  • C.Y. Chen et al.

    Antibody against N-terminal domain of phospholipid scramblase 1 induces apoptosis in colorectal cancer cells through the intrinsic apoptotic pathway

    Chem. Biol. Drug Des.

    (2014)
  • Y. Chen et al.

    Characterization of soluble non-covalent complexes between bovine serum albumin and β-1, 2, 3, 4, 6-penta-O-galloyl-D-glucopyranose by MALDI-TOF MS

    J. Agric. Food. Chem.

    (2004)
  • Y. Chen et al.

    Anticancer efficacy enhancement and attenuation of side effects of doxorubicin with titanium dioxide nanoparticles

    Int. J. Nanomed.

    (2011)
  • A. Dag et al.

    Polymer–albumin conjugate for the facilitated delivery of macromolecular platinum drugs

    Macromol. Rapid Commun.

    (2015)
  • J.F. Deeken et al.

    The blood-brain barrier and cancer: transporters, treatment, and Trojan horses

    Clin. Cancer Res.

    (2007)
  • N. Dwivedi et al.

    Dendrimer-mediated approaches for the treatment of brain tumor

    J. Biomater. Sci. Polym. Ed.

    (2016)
  • J. Fernandes et al.

    Amino acid conjugated chitosan nanoparticles for the brain targeting of a model dipeptidyl peptidase-4 inhibitor

    Int. J. Pharm.

    (2018)
  • R. Ghanghoria et al.

    Luteinizing hormone-releasing hormone peptide tethered nanoparticulate system for enhanced antitumoral efficacy of paclitaxel

    Nanomedicine

    (2016)
  • Cited by (95)

    • Potential theranostic targets in glioblastoma

      2023, New Insights into Glioblastoma: Diagnosis, Therapeutics and Theranostics
    • Toxicity of dental materials and ways to screen their biosafety

      2023, Essentials of Pharmatoxicology in Drug Research: Toxicity and Toxicodynamics: Volume 1
    • Computer-aided technologies in drug discovery and toxicity prediction

      2023, Essentials of Pharmatoxicology in Drug Research: Toxicity and Toxicodynamics: Volume 1
    • Predicting toxicity from chemical structure of a drug compound

      2023, Essentials of Pharmatoxicology in Drug Research: Toxicity and Toxicodynamics: Volume 1
    • Toxicant-induced injury and tissue repair

      2023, Essentials of Pharmatoxicology in Drug Research: Toxicity and Toxicodynamics: Volume 1
    View all citing articles on Scopus
    1

    Authors having an equal contribution and can be interchangeably written as first authors.

    View full text