Nanoparticle Brain Delivery: A Guide to Verification Methods

Figures & data

Figure 1. Brain vasculature and components of the blood–brain barrier/neurovascular unit in relation to brain parenchyma.

Table 1. Studies that reported the brain level of the nanoparticle or component(s) at multiple times after convection enhanced delivery. Mean residence time values were determined as described in nanoparticle kinetics in the brain.

Study summary	MRT (h)	Ref.
The percent of radioactivity in the brain following CED of 82 nm ^3H-cholesteryl hexadecyl ether liposomes was determined	22	[83]
The rat brain hemisphere that received CED of 0.06, 0.8 or 1.6 mg of 96–101 nm irinotecan-loaded nanoliposomes was perfused and homogenized, centrifuged,and irinotecan analyzed by the HPLC. Half-life of the three doses was 6.7, 10.7 and 19.7 days, respectively	108, 181 and 400	[81]
Brains from rats that received CED of 105–122 nm topotecan-loaded liposomes was perfused, the infused hemisphere removed, homogenized, centrifuged and topotecan analyzed by the HPLC. Half-life was 1.5 days	44	[84]
The rat brain hemisphere that received CED of a mixture of 95–110 nm doxorubicin-loaded pegylated liposomes or irinotecan-loaded nanoliposomes was perfused, homogenized, centrifuged and irinotecan and doxorubicin analyzed by the HPLC. Half-life was 10.9 and 16.7 days, respectively	273 and 305	[79]
Local retention of radioactivity after intratumor CED of 135 nm ^186Re-loaded liposomes was determined in rat brain by gamma camera. Half-clearance time was 54.6 h	50	[85]
Brain MRI was obtained after CED of human serum albumin-, PEG- and methotrexate/human serum albumin-coated 20 nm γ-Fe2O3 NPs	190, 184 and 198	[80]
IVIS imaging was conducted after CED of EGFR-coated quantum dot-containing immunoliposome hybrid NPs to mice with brain tumors	97	[86]
Positron emission tomography imaging was conducted after CED of 71 and 147 nm [^18F]-fluorobenzylamine, NHS-PEG- biotinylated, avidin-modified PLGA NPs to rats. Half-life was 111 min	0.39	[87]
Rat brain containing the region of CED of 175 nm carboplatin-labeled fluorescein-PLGA NPs was homogenized and platinum determined by ICP–MS	18	[88]
The hemisphere that received CED of 60–100 nm radiosensitizer (VE822)-loaded PEG-conjugated, poly(u-pentadecalactone-co-p-dioxanone) NPs, of varying PEG polymer ratios, was analyzed by LC–MS for VE822. Half-life was 12 h, 2 and 5 days	46, 61 and 68	[89]
Fluorescence images of mice brain sections were obtained after intrastriatal CED of Cy5.5 fluorophore-labeled peptide-based nanofibers (5-nm width, 100, 400 and 1000 nm length)	20, 25 and 29	[29]
MRI imaging was conducted for 48 h after CED of 54-nm iron oxide chitosan-PEG NPs modified with a glioma-targeting ligand to mice-bearing brain tumors. Signals near the point source of the infusion of NP without and with ligand were used to calculate the MRT	3.4 and 4.8	[90]

CED: Convection-enhanced delivery; HPLC: High-performance liquid chromatography; ICP–MS: Inductively coupled plasma mass spectrometry; IVIS: In vivo imaging system; LC–MS: Liquid chromatography-mass spectrometry; MRT: Mean residence time to the last sample; NHS: N-hydroxysuccinimide; NP: Nanoparticle; PEG: Polyethylene glycol; PLGA: Poly(D,L-lactide-co-glycolide).

Table 2. Visual methods that might be used to image brain nanoparticle distribution and their reported resolution.

Method	Resolution	Ref.
High-resolution brain MRI	≥120 μm	[91]
Photoacoustic microscopy	20–100 μm	[37]
IVIS spectrum	20 μm	[92]
MSI	Submicron for some methods	[93]
Super resolution (light) microscopy (aka: single-molecule localization microscopy)	<100 nm	[38,94–96]
Hyperspectral imaging	<10 nm	[44,97,98]
CLSM	2 μm	[99]
Electron microscopy	<1 nm	[100]
CLEM	>50 nm for LM, <1 nm for EM	[59,61]

CLEM: Correlative light and electron microscopy; CLSM: Confocal laser scanning microscopy; EM: Electron microscopy; IVIS: In vivo imaging system; LM: Light microscopy; MSI: Mass spectrometry imaging.

Table 3. A classification of results demonstrating brain versus. brain parenchymal entry.

Level	Description of results	Interpretation
1	NP component (e.g., fluorescent dye, element) in bulk brain or ex vivo brain not perfused of blood. Determined by methods such as IVIS, radioactivity, NAA, ICP–MS, AAS or HPLC	A NP component was in bulk brain
2	NP component (e.g., fluorescent dye, element) in bulk brain or ex vivo brain perfused to remove blood. Determined by methods such as IVIS, radioactivity, NAA, ICP–MS, AAS or HPLC	A NP component was in bulk brain, but not in the blood within the brain
3	Intact NP in bulk brain not perfused of blood. Determined by methods such as bulk brainhomogenization, NP isolation and verification (e.g., visual, perhaps also requiring chemical)	The NP was in bulk brain
4	Intact NP in bulk brain perfused to remove blood. Determined by methods such as bulk brain homogenization after perfusion to remove blood, NP isolation and verification (e.g., visual, perhaps also requiring chemical)	The NP was in bulk brain, but not in the blood within the brain
5	NP component isolated from brain parenchyma. For example, using the capillary depletion method of brain perfused to remove blood to obtain brain parenchyma and a method to identify the NP component, for example, radioactivity, ICP–MS, AAS or HPLC. For example, microdialysis to recover an NP component from brain extracellular fluid, in the presence of an intact BBB	A NP component was in brain parenchyma
5	NP component (e.g., fluorescent dye, element) visualized in brain areas or cells not associated with brain vasculature. Determined by methods such as MSI, single-molecule localization microscopy, hyperspectral imaging or CLSM	A NP component was in brain parenchyma
6	Intact NP isolated from brain parenchyma. For example, using the capillary depletion method of brain perfused to remove blood to obtain brain parenchyma and a method to verify intact NPs (e.g., visual, perhaps also requiring chemical)	The NP was in brain parenchyma
6	Intact NP visualized in brain areas or cells not associated with brain vasculature. Determined by methods such as TEM, with chemical verification, such as EDS or EELS	The NP was in brain parenchyma

The level of demonstration of nanoparticle brain or brain parenchymal entry is in the first column.

AAS: Atomic absorption spectroscopy; BBB: Blood–brain barrier; CLSM: Confocal laser scanning microscopy; EDS: Energy dispersive x-ray spectroscopy; EELS: Electron energy loss spectroscopy; HPLC: High-performance liquid chromatography; ICP–MS: Inductively coupled plasma–mass spectrometry; IVIS: In vivo imaging system; MSI: Mass spectrometry imaging; NAA: Neutron activation analysis; NP: Nanoparticle; TEM: Transmission electron microscopy.

Table 4. Brain nanoparticle distribution study claims, summary, critique, support for the claims and level of brain or brain parenchymal entry.

Claim	Study summary	Critique, support for claim and level of demonstrated NP brain entry	MRT (h)	Ref.
‘Uptake of NPs in the brain’	Whole organs were solubilized for radioactivity determination after iv. injection of 131-nm surfactant-coated poly(^14C-methyl methacrylate) particles	Claim assumes intact NP distribution to the brain, which is not shown. Bulk brain ^14C presence shown; 1	42 (47)	[101]
‘Doxorubicin was in brain tissue’	Whole organs were homogenized, proteins precipitatedand doxorubicin determined by HPLC after iv. injection of 90-nm doxorubicin-loaded solid lipid particles	HPLC results of NP component show bulk brain uptake; 1	1.8 (1.8)	[102]
‘Polysorbate 80-coated polybutylcyanoacrylate NPs could be used to overcome blood–brain barrier’	Visible blood vessels were removed from the cerebrum and cerebellum, whole organs were homogenized, proteins precipitated and methotrexate determined by HPLC after iv. injection of 70–345 nm methotrexate-loaded PBCA NPs	HPLC analysis of NP component in bulk brain does not demonstrate distribution across the BBB; 1	1.8 and 1.9	[103]
‘ICP–MS results indicated that 17 and 37 nm [gold NPs] were capable of passing through the blood–brain barrier and entering the mouse brain.’	^aWhole brain was homogenized, separated by HPLC,and gold quantified by ICP–MS after ip. injection of 17- and 37-nm gold particles. ^bTEM and EDS showed electron-dense particles in the cytoplasm of hippocampal pyramidal cells and gold	^aICP–MS results of NP component in bulk brain do not demonstrate distribution through the BBB; 1. ^bTEM/EDS results demonstrate distribution across the BBB; 6	Increased from 336 to 504 h after injection	[104]
‘Brain delivery’	Whole brain from transcardially perfused rats was homogenized for gold quantitation by NAA after ip. injection of peptide-conjugated,citrate-coated 12-nm gold particles.Whole brain from transcardially perfused rats was prepared for fluorescence microscopy after ip. injection of carboxyfluorescein-labeled,peptide-conjugated, citrate-coated 12-nm gold particles	NAA results of NP component in brain homogenate and concurrent fluorescence show bulk brain uptake; 2	No consistent increase or decrease over 24 h	[105]
‘Focused ultrasound and magnetic targeting synergistically delivers therapeutic MNPs across the blood–brain barrier to enter the brain’	^aWhole brain was prepared for ICP–OES iron and HPLC epirubicin determination after iv. injection of 12-nm magnetic NPs into rats whose BBB had been opened by iv. injection of microbubbles and focused ultrasound. ^bTEM of similarly treated rats with brain tumors showed electron densities inside tumor cells and macrophages	^aICP–OES and HPLC demonstrate bulk brain but not parenchymal entry; 1. ^bTEM demonstrates delivery across the BBB in the presence of BBB opening, assuming the electron densities are iron; 6	11	[106]
‘NP deposits were widely distributed throughout brain, with apparent cellular uptake’	^aMicroscopic assessment of sections from rat brain after intranasal dosing of 299-nm fluorescent- and ovalbumin-loaded cationic liposomes revealed the label in the striatum and substantia nigra. ^bProtein immunohistochemistry of perfused rat brain demonstrated protein and tyrosine hydroxylase co-localization in striatum and substantia nigra. ^cGamma counting of whole brain after intranasal dosing of 50 μg ^111In-labeled NP in 1 or 2 μg/μl suggested approximately 0.2% of the dose was in the brain, assuming stability of the ^111In-NP	^aImage shows increased fluorescence within cells; 5. ^bColocalization shows fluorescence from NP within cells; 5	^c4.3 and 2.1	[36]
‘[Curcumin-loaded PLGA NPs] could cross the blood–brain barrier (BBB) to enter brain tissue’	Brains from perfused rats were dissected into parts, homogenized,proteins precipitated and curcumin determined by HPLC after iv. injection of 163-nm curcumin-loaded PLGA NPs Reported MRT was 27 min	HPLC analysis of NP component in bulk brain does not demonstrate distribution across the BBB; 2	0.36 (0.33)	[107]
‘In vivo brain distribution … monitored by a real-time fluorescence imaging system’	The whole rat and ex vivo brain were bioluminescence imaged using IVIS and fluorescence microscopic images obtained of brain sections after iv. injection of 190–210 nm fluorescent dye-labeled solid lipid particles	Imaging demonstrates bulk brain distribution; 1	0.45	[108]
‘Brain delivery’	Fluorescence intensity was determined in ex vivo organs from perfused rats after intranasal dosing of odorranalectin-conjugated fluorescent-labeled 90-nm PEG-PLGA NPs	Fluorescence intensity of brain demonstrates bulk brain delivery; 2	5.2	[109]
‘We observed … a fourfold increase of silver content … which indicates that AgNPs are able to cross the blood–brain barrier (BBB)’ ‘AgNPs also gained access to the brain … visible as single NPs as well as clusters’	^aBrain from perfused rats was digested and silver determined by ICP–MS after iv. 20- and 200-nm silver NP injection. ^bTEM showed electron-dense particles and clusters of particles in brain cells. Their elemental composition was not reported	^aICP–MS results of NP component in bulk brain does not demonstrate distribution across the BBB; 1. ^bNPs crossed the BBB if the electron dense particles contained silver that was not the result of dissolution then precipitation; 6	Increased from 24 to 672 h after injection	[110]
‘NPs were localized in the brain parenchyma’	^aGold in brains from perfused rats was determined by INAA after ip. injection of transferrin receptor targeting and β-amyloid destroying peptides conjugated to 37-nm gold NPs. ^bTEM of hippocampus and cortex of perfused rats after ip. injection of these gold NPs demonstrated intracellular electron dense spots	^aINAA results of NP component in bulk brain do not demonstrate distribution across the BBB; 1. ^bNP seen in brain parenchyma; 6, assuming the spots were gold NPs	8.8	[111]
‘Suggest that the OX-26-coated NPs were taken up by the brain’	Microdialysis samples were obtained from the ventral hippocampus for fluorescence detection of FITC-dextran after iv. administration of 140–160 nm CD71/OX26-coated FITC-dextran loaded methoxy-PEG-poly(lactic acid) NPs	NPs were too large to diffuse through the microdialysis membrane suggesting FITC-dextran release; 5, assuming intact BBB	21	[74]
‘Brain endothelial uptake and possibly parenchymal accumulation’	CLSM of fixed brain sections after carotid artery (in situ brain) perfusion and washout of perfusate or iv. injection of fluorescent dye-labeled, polyethylenimine/oligonucleotide-containing liposomes showed fluorescence associated with BMECs and in the perivascular space between the BMEC membrane and parenchymal basement membrane	NP or a component seen beyond BMECs but not in brain parenchyma; 2		[51]
‘NPs possess an appreciable BBB penetrability’	Microdialysis samples were obtained from the right ventrolateral periaqueductal gray for fluorescence detection of FITC-neurotoxin after intranasal dosing of 145-nm chitosan-modified PLA FITC-neurotoxin-containing NPs	NPs were too large to diffuse through microdialysis membrane suggesting FITC- neurotoxin release; 5, assuming intact BBB	3.3	[75]
‘Accumulation was achieved in the brain’	Whole brains from mice were homogenized, proteins precipitated, and camptothecin determined by HPLC after iv. injection of 160 nm camptothecin-containing solid lipid NPs.	HPLC analysis demonstrates bulk brain accumulation; 1	7.7 (7.0)	[112]
‘[Palmitoyl dalargin] was detectable … in the brain’ ‘pDal is clearly located in the brain parenchyma’	^aWhole brains were homogenized, proteins precipitated and palmitoyl dalargin measured by LC-MS in rats after iv. injection of palmitoyl dalargin fibers. The authors claim brain parenchymal entry because brain concentration was greater than can be accounted for by blood, and slower clearance from brain than blood. ^bCoherent anti-Stokes Raman scattering microscopy revealed pDal fibers within brain parenchyma	^aLC–MS of NP component in bulk brain does not rule out BMEC adsorption or incorporation; 1. ^bMicroscopy demonstrated brain parenchymal fiber entry; 6	0.86 (0.17)	[113]
‘Ag detected in the brain and passing through the blood–brain barrier (i.e., not localized in the vessels and capillaries) constitutes no less than 93% of the total content of Ag in the organ’	Silver and gold were determined by NAA in brain of exsanguinated rats after 14 or 28 daily intragastric administrations of 35-nm silver and gold NPs. The authors calculated brain metal from its concentration in blood and brain blood mass	This does not account for metal in BMECs. Silver could be ion released from NP dissolution. BBB Ag passage not shown; 2		[14]
‘In vivo studies indicated BBB penetration of intravenously injected TIMP-1 NPs + Ps80’	Confocal microscopy (40× objective) immunohistochemistry of brain slices from perfused mice after iv. injection of polysorbate 80-coated coumarin 6-containing protein-loaded PLGA NPs showed a few positive spots	Spot location (intra- vs extravascular) cannot be determined. BBB penetration not demonstrated; 2		[114]
‘BBB-penetrating cancer cell-targeting delivery system’	^aWhole animal and ex vivo brain IVIS imaging were conducted after iv. 85-nm fluorescent-labeled, doxorubicin-loaded desoctanoyl ghrelin and folate conjugated-polymersome administration to brain tumor-bearing mice. ^bBrains from brain tumor-bearing perfused rats were homogenized, proteins precipitated and doxorubicin determined by HPLC after iv. injection of 85-nm fluorescent-labeled, doxorubicin-loaded desoctanoyl ghrelin and folate-conjugated polymersomes	^aWhole brain results using this method do not have sufficient resolution to demonstrate BBB penetration; 1. ^bHPLC analysis of NP component in bulk brain does not demonstrate BBB penetration; 2		[115]
‘Targeted ^68Ga-GNPs improves BBB crossing’	Whole brains from perfused rats were gamma counted after iv. injection of glucose conjugate-coated-^68Ga-gold NPs. Brain gold content was determined by ICP–MS	Whole brain radioactivity and ICP–MS results of NP component in bulk brain do not verify BBB crossing; 2		[11]
‘Brain delivery’	Whole organs were homogenized and Bacoside-A determined by HPLC after ip. administration of 173-nm polysorbate 80-coated, Bacoside-A-loaded PLGA NPs to rats	HPLC analysis of NP component demonstrates bulk brain delivery; 1		[116]
‘Brain bioavailability of breviscapine from the Bre-GBSLN-PS and Bre-GBSLN-PS-DSPE was significantly enhanced’	Fifteen kDa cutoff (∼5 nm) microdialysis probes were used to obtain dialysate from rat brain after iv. injection of 23-nm PEG, stearate, 1,2-distearoyl-snglycero-3-phosphoethanolamine-N-PEG2000-coated breviscapine-loaded solid lipid NPs. The primary active component of breviscapine was measured by HPLC	NP released component does not identify NP brain location; 5, assuming intact BBB	No decrease from 3 to 12 h after injection (0.61)	[76]
‘We … demonstrated the ability of these [gelatin NPs] to pass into the brain parenchyma following intranasal administration’	SEM of rat brain 6 h after intranasal administration of 184-nm gelatin NPs showed clusters of approximately 40-nm spherical particles in the olfactory tract, cortex, striatum, amygdala and hypothalamus as evidence of NP brain parenchymal entry	Assuming the observed particles were gelatin NPs, perhaps smaller due to tissue processing, the results suggest brain parenchymal entry; 5		[117]
‘Migration to brain parenchyma’ ‘albumin modulates the transport across the blood–brain barrier’	^aWhole brain radioactivity was determined by gamma-scintigraphy of isolated mouse brain after iv. albumin-coated 15-nm ^198Au NP injection. ^bBrain confocal microscopic images were obtained after iv. fluorescent-labeled albumin-coated ^198Au NP injection	^aWhole brain radioactivity does not verify transport across the BBB into brain parenchyma; 1. ^bResolution is not sufficient to differentiate vascular from brain parenchymal distribution; 1	21 (5.1)	[17]
‘Particles that actually crossed the BBB’	Gold was determined by flame AAS analysis in brain from mice that received iv. injection of 20-nm insulin PEG-coated gold particles. Brain gold was calculated from its blood concentration and brain–blood volume	This does not account for gold associated with BMECs that the authors hypothesize accounted for gold in the brain at late time points; 1	11 (blood did not decrease over 48 h)	[118]
‘Coumarin distribution to the glioma region’	^aBrains from glioma-bearing mice were homogenized, coumarin-6 extracted and quantified by HPLC after iv. injection of 94-nm coumarin-6, L-13Rα2 ligand-coated PEG-PLGA NPs. ^bFluorescence spots were seen in the brain	^aHPLC analysis of NP component demonstrates bulk brain distribution; 1. ^bMicroscopy resolution only sufficient to demonstrate brain distribution; 1		[119]
‘Brain uptake’	Radioactivity determined by gamma-scintigraphy of isolated whole mouse brain after iv. injection of 9- and 40-nm diameter, 396- and 268-nm length [^111In]-DTPA-multiwalled carbon nanotubes	Radioactivity demonstrates distribution of NP or component in bulk brain; 1		[120]
‘Gold would presumably not be cleared from brain in such a short timeframe if it has entered the brain parenchyma’	Brains from mice were digested and gold determined by ICP–OES after iv. injection of 80-nm biodegradable polymeric micelles with 5 nm gold NP cores	ICP–OES results of NP component in bulk brain do not show brain parenchymal entry; 1	43 (7.7)	[78]
‘Demonstrated that the NPs could cross the BBB’	CLSM images were obtained of brain tissue cryosections, stained with tomato lectin to label blood vessels, from rats that received iv. microbubbles and focused ultrasound to open the BBB before iv. injection of 177-nm fluorescent dye-containing PBCA NPs	Fluorescent spots attributed to the fluorescent dye near the blood vessels suggest NPs crossed the BBB in the presence of opened BBB; 6		[47]
‘The peptide was able to diffuse outside the BBB, while NPs were restricted to the vessel endothelium’	CLSM images of brain sections with antibody-stained BMECs from mice iv. dosed with 35-nm fluorometric dye-labeled amphiphilic polymer-coated iron oxide NPs containing a fluorometric dye-labeled antiretroviral drug enabled distinction among microvessels, NPs and drug. The NPs and BMECs were co-localized. The antiretroviral drug was seen within and outside of the vascular compartment, in brain parenchyma	Peptide fluorescence was seen in brain parenchyma; 5		[24]
‘Distributed into the brain tissue’	The capillary depletion method was used to obtain a non-BMEC fraction of mouse brains for LC–MS/MS curcumin analysis after oral dosing of 139, 312 and 412 nm uncoated, chitosan- and N-trimethyl chitosan-coated, curcumin-loaded solid lipid NPs	Most of the whole brain curcumin was in the supernatant fraction, not associated with BMECs, but could have come from blood; 1		[71]
‘BBB penetration by AuNPs’	^aGold content was determined by ICP–MS in brain from perfused rats that received ip. injection of 1.3-nm gold NPs. ^bGold was determined by laser ablation ICP–MS in brain sections	^aICP–MS results of NP component in bulk brain do not verify BBB penetration; 2. ^bLA-ICP–MS resolution not sufficient to verify BBB penetration. 2	8.7 (4.4)	[12]
The increase of brain iron from days 1 to 7 ‘suggested the smaller IOMNs crossed the blood–brain barrier’	Iron was quantitated by AAS in solubilized whole organs from mice that received iv. 10-nm carboxyl-coated iron oxide magnetic NPs	AAS results of NP component in bulk brain do not verify BBB crossing; 1	Increased from 24 to 168 h after injection	[121]
‘Exosome delivered anticancer drugs crossed the BBB and entered into the brain’	Fluorescent dye-, paclitaxel- or doxorubicin-labeled mouse-derived <50 nm BMEC exosomes were iv. injected into zebrafish transgenic for vasculature GFP expression	CLSM suggests NP fluorescence and shows drug fluorescence outside of the vasculature; 5		[48]
‘Nose-to-brain transport’	Cyclosporine A was extracted from homogenized olfactory bulb and hind brain samples and quantitated by LC–MS/MS after intranasal dosing of 272-nm nanoemulsion to the rat	LC–MS/MS NP component shows bulk brain distribution; 1	2.4 and 2.6	[122]
‘NPs were capable of penetrating cell membranes and entering cellular compartments’	Brains from mice with green fluorescent protein-expressing tumors were imaged using CLSM after iv. injection of 165-nm fluorescent dye-labeled poly(amineco-ester) terpolymer NPs. PECAM (in BMECs) was labeled. NPs were visualized around tumor blood vessels and within a cell	Brain parenchymal NP entry shown in tumor cells; 6		[49]
‘Brain accumulation at the site of injury’	Whole-body IVIS images were obtained after iv. administration of approximately 170-nm fluorescent-labeled siRNA-containing biodegradable mesoporous silica, encapsulated in graphene oxide nanosheets. Fluorescence was seen in the hemisphere of mice with damaged brain	Greater fluorescence in brain at the injury site supports brain accumulation; 1		[123]
‘NP was obviously accumulated in brain’	Imaging demonstrated mouse whole brain fluorescence after iv. injection of approximately 180-nm cyclic peptide-modified fluorescence dye-loaded PEG-poly(lactic acid) NPs	Fluorescence demonstrates brain accumulation; 1		[124]
‘Delivery of … NPs across the blood–brain barrier’	CLSM images of mouse brain after oral administration of 60-nm PEG-coated, curcumin and piperine-coloaded glyceryl monooleate NPs showed intracellular NPs	Neuronal intracellular NPs supports delivery across the BBB; 6	18 (13)	[50]
NP were ‘taken up into the brain cells’	Rats with BBB opened by focused ultrasound received iv. 22-nm fluorescent probe-labeled low-density lipoprotein NPs. Fluorescence microscopy images were interpreted as showing punctate fluorescent staining within brain cell cytosol	Brain parenchymal NP uptake appears to be shown in the presence of an opened BBB; 6		[46]
‘NP retention in brain tumors’	^aIVIS-imaged whole and ex vivo mouse brain showed accumulation in glioma after iv. 33–36-nm fluorescent-labeled, penta-amino acid- or 2-cyano-6-amino benzothiazole-, or both, coated gold NP injection. ^bGlioma were excised for gold quantification by ICP–MS. ^cTEM images showed intracellular densities	^aWhole brain results using this method show NP retention in brain tumors; 1. ^bICP–MS NP component in bulk brain shows brain component retention; 1. ^cTEM showed brain parenchymal NP entry in tumor cells; 6		[125]
‘[Quetiapine fumurate]-NP (IV) may have crossed BBB by opening tight junctions’	Brains removed from rats were homogenized and quetiapine quantified by HPLC after iv. injection of 131-nm quetiapine fumarate-loaded chitosan NPs	HPLC results of NP component in bulk brain do not verify BBB crossing; 1	1.5 (1.4)	[126]
‘Successfully overcame the in vivo BBB and BBTB delivery barriers’	^a Ex vivo brain and dissected tumors from mice with brain tumors were fluorescent imaged 4 h after iv. injection of fluorescent dye-loaded cyclic peptide-containing approximately 100-nm liposomes. ^bFluorescent images of brain with fluorescent-stained microvessels show fluorescence from NPs separate from microvessels	^aWhole brain and tumor fluorescence does not demonstrate delivery across the BBB or BBTB; 1. ^bSpots of NP fluorescent dye distinct from blood vessels suggest NP distribution outside of brain vasculature; 5		[127]
‘Ex vivo CT imaging and analysis indicated that INS-GNPs transported across the BBB’	^a In vivo and ex vivo CT mouse brain imaging was conducted after iv. 20-nm insulin-coated, PEG-layered, gold NP administration. ^bFlame AAS of gold from digested brain was determined	^aWhole brain CT images do not verify transport across the BBB; 1. ^bAAS results of NP component in bulk brain do not verify transport across the BBB; 1	15 (18)	[128]
‘Brain microvessel endothelial cell targeting’	Brains of mice were perfused and prepared for immunohistochemistry and fluorescence imaging after 2 iv. injections 1 h apart of 50-nm transferrin antibody-conjugated mesoporous silica NPs. BMECs were labeled with biotinylated potato lectin detected by fluorescent-labeled streptavidin. One hour after the second injection NPs were associated with the BMECs. There was no evidence brain parenchymal penetration	NP brain microvessel targeting was demonstrated; 3		[129]
‘Enhanced drug transport across BBB’	^aIron was determined by AAS in mouse organs after iv. injection of 80-nm dextran-spermine superparamagnetic-core NPs and 96-nm transferrin-coated NPs ^bTissues were stained with Prussian blue to visualize iron localization	^aAAS results of NP component in bulk brain do not verify transport across the BBB; 1 ^bResolution not sufficient to confirm iron transport across the BBB; 1	Increased from h 24 to 168 after injection	[130]
‘To further confirm the translocation of CLS-PEG NPs across the BBB’ ‘C6 to pass through the blood–brain barrier’	^aRat brains were analyzed by fluorescence analysis after iv. injection of 176-nm coumarin-6-labeled cholesterol-containing PBCA NPs and 185-nm particles also containing PEG. ^bBrains from perfused rats were viewed using CLSM that showed uniform extracellular fluorescence	^aFluorescence results do not confirm BBB translocation; 1. ^bNP component in brain extracellular space supports claim of its passage through the BBB; 5	2.2 and 2.3 (3.4 and 5.5)	[52]
‘The NPs crossed the BBB and entered the brain parenchyma’	^aGold in brains from perfused rats was determined by INAA after iv. injection of rhodamine-PEG-angiopep-2-labeled gold nanorods. ^bBrain from a rat was fluorescent imaged after iv. injection of rhodamine-PEG-angiopep-2-labeled gold nanorods. ^cTEM of hippocampus of a perfused rat after iv. injection of PEG-angiopep-2-labeled gold nanorods demonstrated intracellular electron dense spots	^aINAA results of NP component in bulk brain does not demonstrate distribution across the BBB; 1. ^bFluorescence results do not confirm BBB translocation; 1. ^cNP seen in brain parenchyma; 6, assuming the spots were gold nanorods		[131]
‘Brain delivery’	Brains from mice were homogenized for LC/MS RU determination after iv. injection of marigold flower extract (RU)-, polysorbate-80-containing lipid polymer hybrid PLGA NPs	LC/MS results of NP component support bulk brain delivery; 1	2.5 (1.2)	[132]
‘Uptake by neuronal cells and appreciable crossing of blood–brain barrier’	Brains from rats were homogenized, solvent extracted and dimethyl fumarate determined by HPLC after oral administration of dimethyl fumarate-containing solid lipid NPs	HPLC results of NP component in bulk brain do not verify neuronal cell uptake or BBB crossing; 1	5.7 (4.7)	[133]
‘Lamotrigine brain uptake’	Lamotrigine was determined by HPLC in homogenized brain after iv. injection of 125-nm lamotrigine-loaded poly-ε-(D,L-lactide-co-caprolactone) NPs	HPLC results of NP component show bulk brain uptake; 1		[134]
‘[DNA-coated gold NPs] can be successfully delivered to specific brain locations across the BBB in mice using focused ultrasound’	Mice were iv. injected with 6–14-nm fluorescent-labeled DNA-coated gold NPs after unilateral microbubble injection and before ultrasound to open the BBB. The effected hemisphere showed more NPs (bright-field and fluorescence microscopy) than the contralateral hemisphere	Resolution was not sufficient to verify delivery across the BBB; 1		[135]
‘This strategy increased NP translocation into brain parenchyma’	CLSM of brain, stained for GFAP to localize astrocyte foot processes, from mice iv. injected with 180-nm ApoE4-coated, fluorescent dye-labeled, lipid, polysorbate-80 NPs showed some fluorescence from the NPs not surrounded by fluorescence from GFAP	Fluorescence from the NP label appears to be in brain parenchyma; 5		[25]
‘Gamma scintigraphic images showed … the radiolabeled formulations successfully crossed BBB’	^aWhole rat gamma scintigraphy was conducted after iv. injection of 24-nm AZT-containing ^99mTc-labeled nanoliposomes. ^bAZT was determined by HPLC in homogenized brain of in situ blood-perfused rats after iv. injection of 24 nm AZT-containing ^99mTc-labeled nanoliposomes	^aWhole rat radioactivity does not verify brain entry or BBB crossing. ^bHPLC results of NP component in bulk brain do not verify BBB crossing 2	6.2 (8.8)	[136]
‘Blood–brain barrier permeability of the NPs was successfully demonstrated by the incorporation of fluorescent dye, Rhodamine B in the NPs’	Brain from rats was homogenized, solvent extracted and Rhodamine B determined by HPLC after ip. injection of 300–400-nm saxagliptin-loaded chitosan-L-valine-based NPs	HPLC results of NP component in bulk brain do not verify BBB permeability; 1		[137]
‘Brain accumulation of curcumin-loaded NPs’	Fluorescence in homogenized brains from perfused rats was determined by HPLC after iv. injection of 46-nm 6-coumarin-loaded oleic acid-conjugated PEG-poly(ε-caprolactone) NPs	HPLC results of NP component show bulk brain delivery; 2		[138]
‘After intravenous administration, fluorescent [solid lipid NPs] were detected in brain parenchyma outside the vascular bed, confirming their ability to overcome the BBB’	Epi-fluorescence microscopy of rat brain 24 h after iv. injection of 278-nm FITC and Brij 78-containing solid lipid NPs showed spherical fluorescence not associated with an endothelial marker	Spherical fluorescence in absence of blood vessel marker fluorescence suggests NP brain parenchymal entry; 5		[139]
‘Ex vivo imaging and semiquantitative assay of the brains demonstrated the brain penetrating behavior of … NPs’	^aWhole mice were multispectral and ex vivo brain CLSM imaged after iv. injection of 148-nm menthol BSA NPs. ^bCLSM and TEM images were obtained of the pineal gland (which lacks a BBB) of rats after iv. 148-nm menthol BSA silver NP injection	^aWhole-body multispectral and CLSM imaging show bulk brain delivery; 1. ^bTEM shows one electron dense spot. Its chemical composition was not reported; 6, assuming Ag in the spot		[53]
‘Bioavailability of quercetin in the brain’	Brains from rats were homogenized, proteins precipitated and quercetin determined by HPLC after oral feeding of 30–50-nm quercetin-coated superparamagnetic iron oxide NPs	HPLC results of NP component show bulk brain delivery; 1		[140]
‘Brain distribution/delivery’	Brains from rats were homogenized, proteins precipitated and rivastigmine determined by HPLC after iv. injection of 145-nm rivastigmine-loaded mesoporous silica NPs	HPLC results of NP component show bulk brain delivery; 1	6.5	[141]
‘Penetration of NPs into brain parenchyma’	Three adjacent sections from isolated guinea pig brains were assessed by silver-enhanced brightfield microscopy, CLSM and TEM after arterial perfusion for 120 min of approximately 8 nm fluorescent-labeled mercaptoundecyl sulfonic acid/octanethiol coated gold/iron core NPs followed by 30-min washout perfusion	CLSM indicates fluorescence surrounding cell nuclei. Optical microscopy shows silver stain surrounding blood vessels. TEM shows electron dense particles on the luminal wall of BMECs and inside brain cells; 6		[22]
NPs ‘passed the BBB, and entered the cytoplasm of vascular endothelial cells, as depicted by the biodistribution studies, flow cytometry analysis and subcellular TEM micrographs.’	^aBrains from perfused mice were homogenized and cells analyzed by flow cytometry 1 h after ip. injection of fluorescent-labeled glucose- or PEG-glucose-coated silica NPs. ^bBrains from perfused mice were CLSM imaged 1 h after ip. injection of fluorescent-labeled glucose- or PEG-glucose-coated silica NPs. ^cBrains harvested from mice were TEM imaged 1 h after ip. injection of fluorescent-labeled PEG-glucose-coated silica NPs	^aFlow cytometry does not reveal cell type or NP localization in, vs on, the cell; 2. ^bCLSM images do not verify brain parenchymal localization; 2. ^cTEM shows electron densities, perhaps in neurons; 6		[54]
‘NP formulation increased BBB transport … in vivo’	^aWhole mouse fluorescence imaging was conducted from 0.25 to 24 h after intranasal dosing of indocyanine green containing transferrin or RVG-coated BSA NPs. ^bOxytocin in CSF was determined by enzyme-linked immunosorbent assay after intranasal dosing of oxytocin-containing RVG-coated BSA NPs	^aWhole body bioimaging does not verify BBB transport; 1. ^bNP component in CSF does not demonstrate brain uptake	7.4	[82]
‘Enhanced delivery of [doxorubicin] across the BBB’	HPLC of dialysates from microdialysis of rat hippocampus after intranasal dosing of 92-nm polysaccharide NP containing doxorubicin showed doxorubicin concentration peak at 30 min	Peak doxorubicin concentration this soon cannot be due to nose to hippocampus neuronal transport. It would be distribution across the BBB. NP component does not demonstrate brain NP uptake; 1	4.2	[77]
‘[NP] rapidly and effectively delivered drugs to the brain via the nose-to-brain pathway’	Mouse brain was IVIS imaged ex vivo and MRI imaged after intranasal dosing of 110- or 143-nm fluorescent and gadolinium-labeled cholera toxin or BSA NP	Whole brain results using these methods show fluorescence and gadolinium in the brain; 1	11.6	[142]
‘NP delivery to the brain’	Ex vivo brain was IVIS imaged after iv. injection of fluorescent-labeled, microbubble complexed, albumin NPs and ultrasound to brain tumor bearing mice	Whole brain results using this method show fluorescence in the brain; 1		[143]
NP ‘transport across the BBB after intravenous administration’	CLSM images are claimed to show 140–200-nm phosphatidic and sialic acid, 5-hydroxytryptamine-moduline modified PLGA NPs in the hippocampus of rats after their iv. injection	No fluorescence attributed to the NPs can be seen		[55]
‘BBB penetration and NP distribution into brain parenchyma’	Epifluorescence was determined in brain regions that did not contain vasculature after iv. injection of 4-nm Angiopep-2, transferrin receptor ligand, endosome escape unit, fluorescent marker containing malic acid NPs in mice whose cerebrovascular was stained. The results were interpreted to indicate that the NPs traversed the BBB and approximately 40% of plasma fluorescence was in the parenchyma with the highest concentration proximal to the vasculature	The methods suggest the fluorescent component of the NP entered brain parenchyma; 5		[144]
‘[NP] crossing intact BBB was … demonstrated in healthy mice’	Confocal microscopy of mouse brain after iv. injection of 126-nm doxorubicin, mitomycin C and internalizing peptide-labeled terpolymer-lipid hybrid NP after iv. injection of Texas-red-dextran to visualize blood vessels showed particulate doxorubicin fluorescence not associated with blood vessels	Punctate fluorescence attributed to doxorubicin not associated with blood vessels suggests brain parenchymal NP entry; 6		[145]

Mean residence time from studies that reported brain and blood (in parentheses) levels at multiple times after dosing. Study multiple end points are designated by ^{a, b, c} before the study summary and critique. The level of demonstrated nanoparticle brain entry is from the classification described in Table 3. The numbers after the sentences in column 3 refer to the level of demonstrated NP brain entry.

AAS: Atomic absorption spectroscopy; BBB: Blood–brain barrier; BBTB: Blood-brain tumor barrier; BMEC: Brain microvascular endothelial cell; BSA: Bovine serum albumin; CLSM: Confocal laser scanning microscopy; EDS: Energy dispersive x-ray spectroscopy; EELS: Electron energy loss spectroscopy; FITC: Fluorescein isothiocyanate; ICP–MS: Inductively coupled plasma–mass spectrometry; ICP–OES: Inductively coupled plasma–optical emission spectroscopy; INAA: Instrumental neutron activation analysis; ip.: Intraperitoneal; iv.: Intravenous; IVIS: In vivo imaging system; MRT: Mean residence time; MSI: Mass spectrometry imaging; NAA: Neutron activation analysis; NP: Nanoparticle; PBCA: Poly(n-butyl cyanoacrylate); RVG: Rabies virus glycoprotein; TEM: Transmission electron microscopy.

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