PAMAM Dendrimer Nanomolecules Utilized as Drug Delivery Systems for Potential Treatment of Glioblastoma: A Systematic Review

Figures & data

Figure 1 Schematic representation of a PAMAM dendrimer with an amine surface. Each successive amide layer branching outward from the core is represented by a new generation (ie, G1, G2, G3, etc.). The internal space of the dendrimer is void and permits encapsulation of cargo.

Notes: Reproduced from Abbasi E, Aval SF, Akbarzadeh A, et al. Dendrimers: synthesis, applications, and properties. Nanoscale Research Letters, 2014;9(1):247.²⁹

Table 1 Dendrimer Shell Modification(s) and Conjugation(s) for the Respective Dendrimer Generation Investigated, Ordered from G1-G7

Investigation	Dendrimer Construct(s)	Dendrimer Shell Conjugation/Modification(s)
Choi et al (2017)^Citation66	G1	PEG-PLGA
Pedro-Hernandez et al (2017)^Citation72	G1 and G2	Ethyphenyl and dodecyl alkyl
Stenstrӧm et al (2018)^Citation65	G1-G4 [2,2-bis(methylol)propionic acid core]	Ammonium trifluoroacetate end groups
Janiszewska et al (2016)^Citation73	G2	Poly-L-lysine
Uram et al (2018, 2019)^Citation78,Citation79	G3	Biotin
Uram et al (2017)^Citation63	G3	Biotin
McNerny et al (2009)^Citation57	G3	c(RGDyK) and biotin
Kaneshiro et al (2009)^Citation58	G3	Poly-lysine
Singh et al (2019)^Citation59	G3.5-G4	Ammine Carboxyl
Bae et al (2019)^Citation60	G4	Phenylalanine, histidine, and arginine
Munro et al (2019)^Citation61	G4	Hydroxyl:amine (90:10)
Wu et al (2018)^Citation77	G4	PEG
Liu et al (2018)^Citation87	G4	TAT PEG
Bae et al (2017)^Citation71	G4	Histidine and Arginine Histidine and Lysine
Bae et al (2016)^Citation70	G4	Histidine and Arginine Histidine and Lysine
Zhang et al (2015)^Citation74	G4	PEG Hydroxyl
Yang et al (2014)^Citation88	G4	Gd-NGO
Bai et al (2013)^Citation89	G4	Arginine
Qiu et al (2018)^Citation62	G5	β-CD moiety Au
Lesniak et al (2016)^Citation90	G5	N/A
Jiang et al (2016)^Citation75	G5	PEG
Zhao et al (2015)^Citation76	G5	PEG
Yan et al (2011)^Citation54	G5	PEG
Ren et al (2010)^Citation55	G5	N/A
Ofek et al (2010)^Citation56	G5	Polyglycerol
Perez et al (2011)^Citation34	G7	N/A

Table 2 Summary of All Investigations for Treatment of Glioblastoma with PAMAM Dendrimer Technology - Transfection of GB Cells and/Or Penetration of GB Tumor with Dendrimer

Investigators	Cytotoxic Cargo	Cell Lines	Results	Cell Viability Measurement	Distribution Analyses
Bae et al (2019)^Citation60	Apoptin	GBL-14	PAMAM-FHR showed improved apoptin gene transfection efficacy vs PAMAM alone. PAMAM-FHR/pJDK-apoptin complex demonstrated apoptosis induction in the GBL-14 cell line with minimal cytotoxicity in control dermal fibroblasts.	[In vitro] EZ-Cytox assay	N/A
Qiu et al (2018)^Citation62	B-cell lymphoma/leukemia-2 (Bcl-2) siRNA VEGF siRNA	U87MG	Bcl-2 and VEGF-siRNA delivery with Au DENPs-β-CD modified G5 dendrimers had high uptake of vector polyplex and gene product silencing capabilities and low cell cytotoxicity.	[In vitro] MTT assay	[In vitro] flow cytometry
Liu et al (2018)^Citation87	KLAK	U87MG	KLAK-TAT-PEG conjugated G4 dendrimers (PKT-S-PEG) demonstrated enhanced U87MG cytotoxicity via mitochondrial apoptosis as well as increased depth of tumor penetration in U87MG spheroids.	[In vitro] JC-1 assay	N/A
Stenstrӧm et al (2018)^Citation65	p42-MAPK siRNA	U87MG C6	G2, G3, and G4 dendrimers successfully complexed the siRNA. G3 and G4 dendrimers showed toxicity in primary neurons only at high concentrations and G2 showed none. G3 and G4 dendrimers were taken up in C6 cell line for p42-MAPK siRNA delivery.	[In vitro] LDH release	[In vitro] Rhodamine fluorescence microscopy
Uram et al (2017)^Citation63	Biotin Pyridoxal	U-118 SCC-15	Increased cellular uptake of biotinylated G3 PAMAM dendrimers for up to 24 hrs of incubation. Cell viability of U-118-MG cells reduced with high sensitivity using biotinylated G3 PAMAM dendrimers with lower normal cell cytotoxicity.	[In vitro] neutral red lysosomal stability assay & XTT assay	[In vitro] FITC confocal microscopy
Jiang et al (2016)^Citation75	Pep-1 (glioma homing peptides)	U87MG	PEGylated PAMAM dendrimers conjugated with glioma homing peptide (Pep-1) for targeting U87MG cells in vitro and U87MG tumor-bearing mice in vivo. Pep-1 conjugated dendrimers were not cytotoxic to U87MG cells in vitro but was localized within U87MG cell lysosomes. Dendrimer construct accumulated in the tumor within tumor-bearing mice.	[In vitro] MTT assay	[In vitro] FITC fluorescence microscopy [In vivo] FITC & Cy5.5 fluorescence microscopy
Zhang et al (2015)^Citation74	Fibrin-binding peptide CREKA	9L	G4 PAMAM dendrimers delivered intracranially for rodent gliosarcoma. Dendrimers distributed throughout tumor within 15 mins with high permeation and perfusion. Dendrimers cleared at a rate of 0.01 μg/g/hr and accumulated in the renal cortex.	N/A	[In vivo] FITC & Cy5.5 fluorescence
Zhao et al (2015)^Citation76	Fibrin-binding CREKA	U87MG	CREKA-conjugated PEGlyated PAMAM dendrimers enhanced fibrin binding capacity of dendrimers and increases uptake into glioma. Nanoparticles also found in kidney, liver, and spleen.	N/A	[In vitro], [In vivo], & [ex vivo] IVIS spectrum imaging
Yang et al (2014)^Citation88	Epirubicin Let-7g miRNA	U87MG	PAMAM dendrimers conjugated with Gd-NGO carried epirubicin and Let-7g miRNA which inhibited cancer cell growth and showed efficient transfection.	[In vivo] FAM-labelled fluorescence microscopy	[In vivo] FAM -labelled fluorescence microscopy
Yan et al (2011)^Citation54	Tumor vasculature targeted cyclic peptides BBB-permeable angiopep-2 peptides	U87MG	G5 PAMAM dendrimers packaged with peptides target integrin on tumor vasculature and LRP receptors on vascular endothelial cells and tumor cells for targeting U87MG GB cells.	[In vitro] MTT assay	[In vitro] & [In vivo] Den-RGD-Angio fluorescence microscopy
Ofek et al (2010)^Citation56	Luciferase siRNA	U87MG	PEI-PAMAM dendrimers delivered luciferase siRNA to silence luciferase gene expression in U87MG cancer cells with high efficacy and low cytotoxicity.	[In vitro] XTT assay	[In vivo] Bioluminescence imaging with mCherry and FITC-labelled luciferase siRNA

Table 3 Summary of All Investigations for Treatment of Glioblastoma with PAMAM Dendrimer Technology - Inhibition of GB Growth and Proliferation

Investigators	Cytotoxic Cargo	Cell Lines	Results	Cell Viability Measurement	Distribution Analyses
Singh et al (2019)^Citation59	Docetaxel	U87MG GL261	Hybrid dendrimer (G4-G3.5) constructs conjugated with docetaxel were increasingly toxic to U87MGMG cells at high concentrations, non-toxic to control cells, and stable in storage over 180 days.	[In vitro] MTT assay & flow cytometry	N/A
Munro et al (2019)^Citation61	Curcumin	GL261	D-Cys-Cur and D-Cys transfection of GL261 cells demonstrated similar anti-inflammatory properties. Treatment with both complexes in mice equally prolonged mouse lifespan. No difference in mouse tumor sizes were found between treatment groups.	[In vitro] MTT assay	N/A
Wu et al (2018)^Citation77	cMBP	U87MG	MET-targeting cMBP peptides (Den-cMBP10) conjugated to G4 dendrimers showed inhibition of U87MG cell proliferation with reduced pMET, pAKT, and pERK1/2 expression levels. Glioma volume size decreased significantly with Den-cMBP10 and median mouse survival increased by 59%.	[In vitro] TUNEL assay	[In vivo] MRI
Uram et al (2018, 2019)^Citation78,Citation79	Celecoxib Fmoc-L-Leucine	U-118 SCC-15 HaCaT	Conjugated dendrimer delivery of both celecoxib and Fmoc-L-Leucine at a ratio of 1:1 showed significant cytotoxicity in U-118 cells. Decreased cell viability, mobility, and proliferation in U-118 cells with administration of a lower dose of G3-BCL (1–2 μM)	[In vitro] NR assay [In vitro] Apo-ONE^®Homogenous Caspase-3/7 Assay	[In vitro] Fluorescence confocal microscopy
Choi et al (2017)^Citation66	Quercetin Acetazolamide	U251	Quercetin conjugated PEG-PLGA micelles induced minimal cell death and reduced nitric oxide release. Acetazolamide conjugated PEG-PLGA micelles showed increased cell death.	[In vitro] Hoechst stain & spheroid cultures	[In vitro] UV-absorbance spectrometry
Bae et al (2017)^Citation71	Apoptin	U87MG	PAMAM-H-R and PAMAM-H-K showed increased transfection efficiency compared to PAMAM. Decreased cell cytotoxicity with PAMAM-H-R/pJDK-apoptin use and greater apoptosis induction and loss of mitochondrial membrane potential than PAMAM-H-K/pJDK-apoptin.	[In vitro] EZ-Cytox cell viability assay, Caspase 3 activity assay, & cell cycle distribution	[In vitro] flow cytometry & fluorescence confocal microscopy
Pedro-Hernandez et al (2017)^Citation72	Ibuprofen	U251 PC-3 K-562 HCT-15 MCF-7 SKLU-7 MDA-MB-231	Ibuprofen conjugated to the resorcinarene-PAMAM dendrimers resulted in high inhibition of cell growth and cytotoxicity in all cell lines. Dendrimer conjugation and modification potency: ethylphenyl group with 16 ibuprofen moieties > dodecyl group with 16 ibuprofen moieties > dodecyl group with 8 ibuprofen moieties > ethylphenyl group with 8 ibuprofen moieties.	[In vitro] Protein-binding dye sulforhodamine B cytotoxic assay	[In vitro] hydrolysis release analysis & fluorescence microscopy
Janiszewska et al (2016)^Citation73	siRNA	U87MG C6	PLL dendrimers protect siRNA from degradation and inhibited cell proliferation of GB cell lines; PLL dendrimers mediate cell toxicity with ROS production after 24 hrs with mitochondrial depolarization to inhibit GB cell proliferation.	N/A	[In vitro] fluorescence microscopy
Bae et al (2016)^Citation70	Apoptin	GBL-14 GBL-37	PAMAM-H-R shows better transfection efficiency and higher expression of apoptin vs PAMAM and PAMAM-H-K dendrimers. 36 hr transfection in tumor cells with PAMAM-H-R/pJDK-apoptin resulted in cell death and no apoptosis in dermal fibroblasts with loss of mitochondrial membrane potential and depletion of cell glutathione levels.	[In vitro] EZ-Cytox cell viability assay kit, LDH assay, & glutathione assay	[In vitro] confocal microscopy & FACS
Lesniak et al (2016)^Citation90	Salicylic acid	U87MG	G5 PAMAM dendrimers conjugated to salicylic acid delivered to U87MG GB-bearing mice. CEST and MRI imaging showed 50% of the tumor images contained the G5 dendrimer construct.	N/A	[In vivo] chemical exchange saturation transfer (CEST) MRI
Bai et al (2013)^Citation89	IFN-β	U87MG	G4 PAMAM-R/pORF dendrimers packaged with IFN-β inhibited cancer cell growth in vitro by 27%. Xenografts of cancer cells with dendrimer construct inhibited cancer cell growth.	[In vitro] MTT assay [In vivo] TUNEL assay	[In vitro] ELISA (GFP and luciferase)
Perez et al (2011)^Citation34	siRNA	T98G J774	G7 dendrimers packaged with siRNA administered with an endocytotic inhibitor. Dendriplexes were taken up by clathrin-dependent endocytosis and caveolin-mediated endocytosis in J774 cells and by cholesterol, caveolin, and actin cytoskeleton pathways in T98G cells.	N/A	[In vitro] Trypan blue and FITC-labelled fluorescence microscopy
Ren et al (2010)^Citation55	Taxol miR-21 inhibitor	U251	PAMAM dendrimers packaged with taxol and miR-21 inhibitors enhanced cell apoptosis in U251 and LN229 (control) cells.	[In vitro] MTT assay [In vivo] annexin V/PI staining and flow cytometry	[In vitro] microscopy of transwell chambers
McNerny et al (2009)^Citation57	Methotrexate	U87MG HUVEC	PAMAM dendrimers conjugated with c(RGDyK) packaged with methotrexate inhibited tumor growth in vitro.	[In vitro] XTT assay	[In vitro] flow cytometry [In vivo] confocal microscopy
Kaneshiro et al (2009)^Citation58	Doxorubicin Luciferase siRNA	U87MG	G3 Poly(L-lysine) dendrimers with a silsesquioxane cubic core and c(RGDfK) conjugate were packaged with doxorubicin and siRNA. Successfully inhibited U87MG-Luc cell growth from doxorubicin-induced cytotoxicity and siRNA effects.	[In vitro] MTT assay	[In vitro] Cy3-siRNA confocal microscopy

Figure 2 A representation of G4-PAMAM-NH2 dendrimer depicting some possible surface modifications on their surface (figure not to scale).

Note: Adapted from Materials Today, 18(10), Kesharwani P, Banerjee S, Gupta U, et al, PAMAM dendrimers as promising nanocarriers for RNAi therapeutics, 565–572, Copyright (2015), with permission from Elsevier.⁹¹

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