Neurons form the basic structural and functional component of the CNS. The primary function of neurons is to respond to stimuli by conducting electrical signals along conductive processes (i.e., axons). The conduction of electrical impulses results in the release of neurotransmitters that further regulate (positively and negatively) nearby neural responses [200]. This enables the brain to maintain a highly complex communication network. A few studies have reported expression of Oatp1a5 and Oatp2b1 in neurons isolated from rat and mouse brain [201]. The neuronal expression of Oatp2b1 is particularly compelling because many of its transport substrates (i.e., prostaglandins and leukotriene C4) are involved in inflammatory signaling and regulation [201]. Because neuroinflammation is a central component of multiple disease states, neuronal Oatp2b1 may represent a viable molecular target for the treatment of CNS diseases. Additionally, Oatp1a5 and/or Oatp2b1 may be involved in neurotoxicity due to their ability to transport cytotoxic microcystins [202]. Confocal microscopy imaging of human frontal cortical tissue demonstrated the expression of OATP3A1 in neurons at both the cell body and axon [169]. In vivo studies have shown that murine neurons express high levels of Oatp2a1 [203]. Additionally, OCT2 has been detected in human neurons [126], while Oct2 has been observed in murine neurons from the amygdala, dorsal raphe, frontal cortex, hippocampus, thalamus, median eminence, and pituitary gland [204,205]. In terms of efflux transporters, P-gp expression has been reported to be negligible in neurons but can be induced in response to pathological stressors [206–208]. In contrast, Mrp1 has been detected in primary cultures of murine prefrontal cortical neurons [209]. While these studies provide evidence for neuronal transporter expression, their role in CNS drug distribution and the effects on treatment effectiveness for neurological diseases requires more extensive evaluation. Such studies are warranted given the possible role of neuronal transporters in controlling drug distribution in the brain parenchyma and/or the access of therapeutic agents to their site of action.

#### **5. Summary and Conclusions**

The challenges of CNS drug delivery are best exemplified by ischemic stroke, which continues to be a significant cause of death and disability in the United States and worldwide. Since FDA-approved pharmacotherapy for ischemic stroke remains restricted to fibrinolytic therapy (i.e., r-tPA), safe and effective neuroprotective drugs are urgently needed. The application of these new chemical entities to the population of stroke patients is highly dependent upon transport across the BBB. Previous research by our laboratory has revealed several endogenous transporters with considerable potential to be targeted for optimization of CNS drug delivery; however, there are few studies in the preclinical stroke literature that describe the need to consider BBB transport mechanisms during therapeutic development. It is striking that statins and memantine, drugs that have shown varying

degrees of success in stroke, are substrates for uptake transporters that are expressed at the human brain microvascular endothelium (i.e., OATP1A2 for statins; OCT1/OCT2 for memantine) while other compounds (i.e., disufenton sodium) that are not transported at the BBB have failed in the clinic [98]. Our laboratory has undertaken important studies to address this critical issue. In the case of memantine, we have been successful by demonstrating that membrane transport mediated by Oct1/Oct2 is a critical step that enables this drug to function as a neuroprotectant in the setting of ischemic stroke [127]. Our work has revealed that the BBB does not simply remain "open" and "leaky" following stroke. Rather, endogenous transporters are still capable of providing selective delivery of their solutes to ischemic brain tissue despite the existence of a route for paracellular diffusion. It is also important to consider that CNS disposition of a single drug is often determined by the combined activity of many different transporters that can move their substrates in opposite directions. Understanding the "multi-transporter environment", both at the BBB and in cellular compartments of the NVU, is essential to moving the field forward by performing detailed and translational preclinical studies that can be developed into effective treatments for neurological diseases. This concept is emphasized by our recent publication where we rigorously evaluated the combined effects of Oatp1a4, P-gp, and Bcrp on the disposition of statin drugs (i.e., atorvastatin, pravastatin, and rosuvastatin) in the brain [49]. Taken together, these findings will facilitate the discovery of treatment strategies where small molecule neuroprotective drugs can be delivered safely and efficiently. Continued research in this area will enable preclinical stroke research to take a "giant step forward" via incorporation of drug transporter experiments into in vivo models of experimental stroke to understand how therapeutics can attain efficacious concentrations in the brain. The future discovery of new chemical entities and/or the development of neuroprotective treatment strategies for ischemic stroke will greatly depend upon obtaining a rigorous understanding of BBB transport mechanisms. Success in this area will also depend upon expansion of transporter studies to include other cell types of the NVU. Indeed, transport mechanisms in astrocytes, microglia, pericytes, and neurons can play a key role in determining drug distribution in the brain parenchyma and, by extension, the capability of a therapeutic agent to reach its site of action. Overall, endogenous transporters at the BBB/NVU represent an untapped opportunity that must be pursued to accelerate the development of pharmacological strategies for the treatment of neurological diseases such as ischemic stroke.

**Author Contributions:** P.T.R. and T.P.D. wrote and edited the original draft of the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work is funded by grants from the National Institutes of Neurological Diseases and Stroke (NINDS; R01 NS084941) and the American Heart Association (19TPA34910113) to PTR and by a grant from the National Institute on Drug Abuse (NIDA; R01 DA051812) to TPD and PTR.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The authors do not have any conflict to declare.

#### **References**

## *Review* **Blood–Brain Barrier Transport of Transferrin Receptor-Targeted Nanoparticles**

**Maj Schneider Thomsen 1 , Kasper Bendix Johnsen 1,2,3, Krzysztof Kucharz 4 , Martin Lauritzen 4 and Torben Moos 1,\***

**Abstract:** The blood–brain barrier (BBB), built by brain endothelial cells (BECs), is impermeable to biologics. Liposomes and other nanoparticles are good candidates for the delivery of biologics across the BECs, as they can encapsulate numerous molecules of interest in an omnipotent manner. The liposomes need attachment of a targeting molecule, as BECs unfortunately are virtually incapable of uptake of non-targeted liposomes from the circulation. Experiments of independent research groups have qualified antibodies targeting the transferrin receptor as superior for targeted delivery of nanoparticles to BECs. Functionalization of nanoparticles via conjugation with anti-transferrin receptor antibodies leads to nanoparticle uptake by endothelial cells of both brain capillaries and post-capillary venules. Reducing the density of transferrin receptor-targeted antibodies conjugated to liposomes limits uptake in BECs. Opposing the transport of nanoparticles conjugated to high-affine anti-transferrin receptor antibodies, lowering the affinity of the targeting antibodies or implementing monovalent antibodies increase uptake by BECs and allows for further transport across the BBB. The novel demonstration of transport of targeted liposomes in post-capillary venules from blood to the brain is interesting and clearly warrants further mechanistic pursuit. The recent evidence for passing targeted nanoparticles through the BBB shows great promise for future drug delivery of biologics to the brain.

**Keywords:** antibody; blood–brain barrier; endosomal; liposome; nanoparticle; targeting; transferrin

#### **1. Introduction**

The brain harbors a vascular barrier system consisting of the blood–brain barrier (BBB) and the blood–cerebrospinal fluid (CSF) barrier. Together, they limit the passage from the bloodstream into the brain parenchyma of virtually any drug unless highly lipophilic or smaller than approximately 70 Daltons [1–4]. This is preferable from a physiological perspective, as it allows to sustain integrity and maintain stable extracellular concentrations of solutes in the brain with minimal influence from fluctuations in blood. It is also advantageous from a toxicologic perspective, as the vascular barriers form a strong defense that prevents the entry of unwanted exogenous substances and pathogens [1–4].

However, the presence of the brain barrier system is inconvenient from a pharmacological perspective as many of the existing and forthcoming drug candidates, e.g., polypeptides, or genetic material-based pharmaceutics like siRNA or cDNA, are unable to pass the brain barriers [5]. This is unfortunate as research of recent years has identified several targets putatively amendable for the treatment of CNS diseases, providing that such biologic therapeutics ("biologics") can enter the brain [6,7]. Consequently, current therapeutics

**Citation:** Thomsen, M.S.; Johnsen, K.B.; Kucharz, K.; Lauritzen, M.; Moos, T. Blood–Brain Barrier Transport of Transferrin Receptor-Targeted Nanoparticles. *Pharmaceutics* **2022**, *14*, 2237. https://doi.org/10.3390/ pharmaceutics14102237

Academic Editor: William M. Pardridge

Received: 24 September 2022 Accepted: 14 October 2022 Published: 19 October 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

attempting to treat neurodegenerative disorders like Alzheimer's and Parkinson's disease, and psychiatric diseases, e.g., schizophrenia and endogenous depression, where the vascular barriers are supposedly intact [8], are pharmacologically restricted.

The discovery of the selective expression of targetable proteins by brain endothelial cells (BECs) has changed the scene and provides new optimism, as several biologics conjugated to targeting antibodies are now amendable for uptake and transport across the BECs [2,5,6]. The use of targeted antibody-conjugated nanoparticles for drug delivery to the brain denotes an interesting alternative to the use of biologics conjugated directly to antibodies [9,10]. A major advantage of nanoparticles is their omnipotence to encapsulate potentially any drug of interest with minimal restraints on their size [9,10]. From the first studies on BBB transport performed more than two decades ago [11–13], the exploration of targeted liposomes and other types of nanoparticles, e.g., polymeric nanoparticles, gold nanoparticles, and magnetic nanoparticles, has progressively continued [9]. A recent peak in this discovery was the identification of transport of transferrin receptor-targeted liposomes into the brain, recently reported using real-time two-photon microscopy in vivo. Interestingly, transferrin receptor-targeted liposomes were found to mainly undergo transport into the brain via an unexpected route, i.e., across endothelial cells of post-capillary venules, and not via brain capillaries [14].

Here, we review the most successful attempts made to enable targeted uptake and transport of nanoparticles across the BBB. As most studies target the transferrin receptor, our delineation of the existing literature mainly addresses this receptor. We describe how specific proteins expressed by BECs enhance the binding and uptake of antibodies from the circulation. Next, we cover how targeted nanoparticles can undergo specific binding and uptake, when conjugated with antibodies weakened in affinity or avidity. We also discuss the therapeutic use of targeted nanoparticles in conditions with brain pathology.

#### **2. Passaging of Large Molecules through the Blood–Brain and Blood–CSF Barriers**

The BBB proper consists of endothelial cells connected by tight junctions. The blood– CSF barrier is formed by choroid plexus epithelial cells also connected by tight junctions, but opposed to the BBB; the capillaries of the blood–CSF barrier are leaky, meaning that solutes of the plasma diffuse into the extracellular space of the choroid plexus where the epithelial cells selectively transport molecules to the CSF [2,3,14]. From a quantitative drug delivery perspective, passaging across the BBB is by far the most important as the brain microvasculature has a surface area thousand-fold higher than that of the choroid plexus. This allows drugs to enter the entire brain while transport across the blood–CSF barrier is restricted to the ventricular system [2,3,15].

The BBB prevents large molecules and particles in blood plasma from entering the brain (Figure 1). This includes entry via the paracellular space between the endothelial cells where tight junctions limit diffusion from blood to brain [1,2,9]. To enable nutrient uptake while preventing the influx of unwanted substances, the BECs express nutrient transporters for, e.g., amino and fatty acids, monosaccharides, vitamins, and essential ions and metals [1–4]. In contrast, the transport of large molecules of the plasma, like albumin and IgG, is diminutive, e.g., intravenous injection of non-immune IgG in the adult rat is limited to as little as 0.03% of the injected dose, which can be surpassed more than ten-fold by injecting anti-rat transferrin receptor-targeted IgG (OX26) [16]. This can also be observed at the ultrastructural level, where a limited number of transporting vesicles occurs in the cytosol of the BECs compared to endothelial cells of other non-fenestrated capillaries, e.g., those of skeletal muscles. For the same reason, the chances of obtaining transport through the brain endothelium of large constituents like nanoparticles are also predictably low, unless nanoparticles are made targetable to nutrient transporters (see next paragraph).

**Figure 1.** Illustration of transport of the three major plasma proteins and targeted anti-transferrin receptor antibodies within brain capillary endothelial cells (BECs). (**A**) Albumin and non-immune IgG enter the BEC non-specifically by fluid-phase uptake. They may undergo release from the BEC at the abluminal side, although the quantitative evidence is scarce and clearly shows that this mean for entry of albumin and non-immune IgG to the brain is negligible. (**B**) Similarly, the transcytotic transport of iron-containing (holo-transferrin) is also negligible and opposed by the release of iron from transferrin due to the lower pH of the endocytosis vesicle. Transport through the BECs may occur to a higher extent in the developing brain where transferrin receptor expression by BECs is far-fold higher than in the adult brain (see body text). (**C**) Transport of high-affine IgG targeted to the transferrin receptor. Transport of this antibody through BECs is negligible. (**D**) Transport of lowaffine IgG targeted to the transferrin receptor. Transport of this antibody through BECs is enabled by the detachment of the antibody from the transferrin receptor in late endosomes, which enables this antibody to undergo transport into the brain. Modified from [15]**. 3. The Transferrin Receptor as Target for Drug Delivery Figure 1.** Illustration of transport of the three major plasma proteins and targeted anti-transferrin receptor antibodies within brain capillary endothelial cells (BECs). (**A**) Albumin and non-immune IgG enter the BEC non-specifically by fluid-phase uptake. They may undergo release from the BEC at the abluminal side, although the quantitative evidence is scarce and clearly shows that this mean for entry of albumin and non-immune IgG to the brain is negligible. (**B**) Similarly, the transcytotic transport of iron-containing (holo-transferrin) is also negligible and opposed by the release of iron from transferrin due to the lower pH of the endocytosis vesicle. Transport through the BECs may occur to a higher extent in the developing brain where transferrin receptor expression by BECs is far-fold higher than in the adult brain (see body text). (**C**) Transport of high-affine IgG targeted to the transferrin receptor. Transport of this antibody through BECs is negligible. (**D**) Transport of low-affine IgG targeted to the transferrin receptor. Transport of this antibody through BECs is enabled by the detachment of the antibody from the transferrin receptor in late endosomes, which enables this antibody to undergo transport into the brain. Modified from [15].

The first indicator of specific uptake and transport of a plasma protein came from observations showing that the brain has a high binding capacity for transferrin, the transporter of the essential metal iron [17]. Later, the identification of a specific binding protein

#### **3. The Transferrin Receptor as Target for Drug Delivery**

The first indicator of specific uptake and transport of a plasma protein came from observations showing that the brain has a high binding capacity for transferrin, the transporter of the essential metal iron [17]. Later, the identification of a specific binding protein for transferrin, the transferrin receptor (a.k.a. transferrin receptor 1), was identified on BECs [18]. Except for a few other organs, e.g., gonadal cells, the expression of the transferrin receptor by BECs is different from capillaries of organs elsewhere in the body, which do not express this receptor [18–23].

Among the large proteins present in blood plasma, transferrin stands out because of its potential for binding to the transferrin receptor of the BECs [9,24]. The quantitative uptake of iron-containing transferrin by the receptor was first addressed thirty years ago in seminal studies by Morgan and co-workers, who co-examined brain uptake of radiolabeled iron together with iodine-labeled transferrin (reviewed in [24]). This allowed for accurate measures of uptake of both iron and transferrin by the brain and, importantly, showed that the transport of radioactive iron through the BBB by far exceeded that of transferrin even a few hours after injection into the peripheral blood. Similar observations were made independently by another research group [25]. This led to the conclusion that iron-containing transferrin is taken up by receptor-mediated endocytosis at the luminal membrane of brain capillaries. In the brain, iron dissociates from transferrin within the slightly acidic environment in the endosomal compartment [26], and iron is transported across the abluminal lipid bilayer of the BECs to the brain, whereas iron-free transferrin is retro-endocytosed back to the luminal membrane [9,24,27].

Counteracting the notion of receptor-mediated endocytosis and retro-endocytosis of transferrin at the BBB, other studies suggested that the iron-containing transferrin may be transferred across the BECs [28,29]. A caveat, in the relevance of these data for understanding iron and transferrin transport at the BBB, shows only transferrin was detected in the brain, hence leaving out the possibility of interpreting the simultaneous transport of iron. However, supporting that transendothelial transport of iron-containing transferrin may occur, observations made on iron and transferrin uptake combined in other studies do not exclude that a minority of iron–transferrin may pass through the BBB, hence simulating transcytosis at the brain endothelium [24].

The expression of transferrin receptor by BECs varies throughout development, with transferrin transport into the brain being higher in the developing brain than at later ages [30–32]. Interestingly, although magnitudes lower than that of iron, the transport of transferrin across the BBB is significantly higher than that of albumin [28–30], which may be due to higher transcellular trafficking of transferrin. Many more vesicles, typically sized about 70 nm in diameter, are present in BECs of the developing brain [33], so even if only a limited fraction of these vesicles fuse with the abluminal membrane, there would a priori be more vesicles emptying their content into the brain during ontogenesis than in adulthood. Endocytic clathrin-coated vesicles are formed as part of transferrin receptor docking at the luminal side of the BECs. The resulting vesicle forming due to the transferrin attachment will likely also capture fluids from the extracellular space of the luminal side in a non-specific manner, which may explain why albumin also gets transferred through the BBB to a higher degree in the developing brain (Figure 1).

Returning to the attempts to enable transport through the choroid plexus, it should not be overlooked that there is strong morphological and physiological evidence for vesicular transport by transcytosis through this epithelium. Ultrastructurally, tracer studies using peroxidases demonstrate that the choroid plexus epithelium can take up large molecules like horseradish peroxidase (HRP) with transfer from the basolateral to the luminal side eventually leading to release into the ventricles [15,34,35]. The choroid plexus, contrary to the brain endothelium, also contains vesicular structures with albumin, IgG, and transferrin. This is corroborated by the concentration of these proteins being many times higher in the ventricular CSF compared to the interstitial fluid of the brain in the CSF (c.f. [15]). The transport of large plasma proteins could theoretically qualify the blood–CSF barrier

as a feasible route for nanoparticles to enter the brain. Counteracting this consideration, CSF of the ventricular system purely distributes substances to the ventricular system and subarachnoid space and excludes the possibility of targeted transport of antibodies and nanoparticles into the brain via transport across the choroid plexus [2].

#### **4. Specific Proteins Expressed by Brain Endothelial Cells Enhance the Binding and Uptake of Antibodies from the Circulation**

The functional capacity of the brain endothelium to bind and internalize antibodies targeted to the transferrin receptor [18] spawned the idea of using transferrin receptor antibodies to target the brain endothelium, as this would allow conjugated therapeutic molecules to enter the brain [19,21,36,37]. The rationale for injecting transferrin receptortargeting antibodies is that exogenous transferrin needs to compete with endogenous transferrin of blood plasma, which significantly reduces the likelihood for binding transferrin receptors [36,37]. This is not the case when using transferrin receptor targeting antibodies, which bind to epitopes at the transferrin receptor without interfering with endogenous transferrin. This is advantageous from the physiological point of view, as the brain delivery of iron is not hampered by antibody-targeting of the transferrin receptor [36,37].

The injection of antibodies targeted to the transferrin receptor dramatically increases the brain uptake as compared to non-targeted antibodies [11,36,37]. Noteworthy, injecting antibodies targeting the insulin receptor, also expressed by BECs, similarly allowed for higher uptake in BECs [11,13,19,38]. However, the internalization did not guarantee successful passage across the endothelium. It was later shown that although transferrin receptor-targeted antibodies were internalized in the BECs by receptor-targeting, the antibodies fell short in their capability to pass to the brain parenchyma [16,39]. This observation was explained by antibodies forming covalent binding to the transferrin receptors sufficient to prevent the antibodies from detaching from the receptor. Later, biotechnological advances created the basis for synthesizing mono-specific antibodies lowered with low affinity, and bi-specific antibodies with low avidity. Such antibodies can be constructed by replacement of a single Fab fragment of a monospecific, high-affine, divalent antibody with a Fab fragment able to bind a different molecule. The properties of the modified antibodies counteracted the permeability restraints of the BBB and enabled both uptake and higher transport of transferrin receptor-targeted antibodies into the brain, as verified from their engagement with neurons and proteins deposited extracellularly in the brain [40,41]. Subsequently, these approaches inspired the generation of a plethora of differently designed transferrin receptor-targeting antibodies, all able to transport conjugated biologics across the BBB [41–47]. Together, they have provided new optimism on how to achieve delivery of therapeutics to the brain, with transferrin receptor-targeted antibodies being now amendable for clinical use in conjugation with enzymes needed for treatment of lysosomal deficiency or being tested in clinical trials against amyloid deposition in Alzheimer's disease (ClinicalTrials.gov Identifier: NCT05371613; NCT04639050; NCT04573023).

While the development of antibodies entering the brain was generated using antibodies targeting the transferrin receptor and the insulin receptor, it should not be overlooked that targeting other proteins of the brain endothelium has been pursued. The large amino acid transporter (CD98hc), glucose transporter 1 (GLUT1), and basigin (CD147) are particularly rich in their selective expression by the BECs compared to capillaries in the periphery, e.g., lung and liver, and are alternatives for targeting the brain endothelium [19,38,48,49].

#### **5. Specific Proteins Expressed by the Brain Endothelium also Facilitate the Binding and Uptake of Targeted Nanoparticles**

The plethora of in vivo studies on nanoparticle transport typically omit to characterize the pharmacokinetics that leads to transport through the BBB. They rather focus on pharmacodynamics or therapeutic effects after the particles have undergone transport into the brain., Often the evidence for the latter is scarce and is extrapolated from pharmacological studies, where improvement in behavioral tasks of experimental animals treated with nanoparticles is used as evidence for BBB transport. Many studies determine fluorescent

nanoparticles using whole-brain imaging, which prevents the distinction of nanoparticles in BECs versus neurons or glia. Other studies determine changes in protein or gene expression by neurons and glia in dissected brain preparations without taking the expression levels in BECs into consideration, e.g., by analyses of brain capillaries isolated and separated from the remaining brain tissue [50]. nanoparticles in BECs versus neurons or glia. Other studies determine changes in protein or gene expression by neurons and glia in dissected brain preparations without taking the expression levels in BECs into consideration, e.g., by analyses of brain capillaries isolated and separated from the remaining brain tissue [50]. In spite of the limited number of studies dealing with the uptake and transport ki-

fluorescent nanoparticles using whole-brain imaging, which prevents the distinction of

*Pharmaceutics* **2022**, *14*, x FOR PEER REVIEW 6 of 19

In spite of the limited number of studies dealing with the uptake and transport kinetics by BEC, a common observation is that the uptake of nanoparticles, e.g., liposomes, gold nanoparticles, or quantum dots, from the circulation is significantly enhanced when conjugated to the transferrin receptor targeted antibodies [51–55]. Comparing the uptake of stealth liposomes in the mouse brain with or without conjugation to proteins putatively targeting BECs, only antibodies targeting the transferrin receptor (clone RI7217) enhanced the liposomal binding and uptake by the brain endothelium [52]. The uptake of RI7217 conjugated liposomes was almost two-fold higher compared to binding with endogenous transferrin or un-conjugated liposomes in brain capillaries isolated from the brain 12 h post injection [52]. Independent studies in the rat [51,53] and mouse [54,55] also concluded that targeting the transferrin receptor using high-affine anti-transferrin receptor antibodies leads to preferential accumulation of liposomes within BECs (Figure 2). Given this evidence, it stands out as somewhat puzzling that reports continuously occur addressing targeting attempts to the transferrin receptor at BECs using only transferrin and not the antibody. netics by BEC, a common observation is that the uptake of nanoparticles, e.g., liposomes, gold nanoparticles, or quantum dots, from the circulation is significantly enhanced when conjugated to the transferrin receptor targeted antibodies [51–55]. Comparing the uptake of stealth liposomes in the mouse brain with or without conjugation to proteins putatively targeting BECs, only antibodies targeting the transferrin receptor (clone RI7217) enhanced the liposomal binding and uptake by the brain endothelium [52]. The uptake of RI7217 conjugated liposomes was almost two-fold higher compared to binding with endogenous transferrin or un-conjugated liposomes in brain capillaries isolated from the brain 12 h post injection [52]. Independent studies in the rat [51,53] and mouse [54,55] also concluded that targeting the transferrin receptor using high-affine anti-transferrin receptor antibodies leads to preferential accumulation of liposomes within BECs (Figure 2). Given this evidence, it stands out as somewhat puzzling that reports continuously occur addressing targeting attempts to the transferrin receptor at BECs using only transferrin and not the antibody.

**Figure 2.** Uptake of fluorescently labeled immunoliposomes conjugated with high-affine IgG targeted to the transferrin receptor (OX26) in brain capillaries in vivo in the rat as revealed using spinning disk confocal microscopy. The OX26 immunoliposomes associate to brain microvessels. Immunohistochemical detection of the OX26 of the immunoliposome similarly reveals that the immunoliposome and its binding antibody accumulate in the brain capillaries. Scale bar = 20 µm. Modified from [53]. **Figure 2.** Uptake of fluorescently labeled immunoliposomes conjugated with high-affine IgG targeted to the transferrin receptor (OX26) in brain capillaries in vivo in the rat as revealed using spinning disk confocal microscopy. The OX26 immunoliposomes associate to brain microvessels. Immunohistochemical detection of the OX26 of the immunoliposome similarly reveals that the immunoliposome and its binding antibody accumulate in the brain capillaries. Scale bar = 20 µm. Modified from [53].

The uptake of RI7217-conjugated liposomes at the BBB is significantly higher compared to stealth liposomes conjugated with non-immune IgG [52]. In contrast, liposomes conjugated to proteins putatively capable of targeting BECs, e.g., (i) cross-reacting material (CRM) with affinity for an endogenous diphtheria toxin receptor; (ii) angiopep-2 with affinity for LRP-1; (iii) COG133 with affinity for apolipoprotein E, all failed to exhibit higher uptake compared to non-immune IgG-conjugated liposomes [52]. In particular, the observations made on liposomes targeting the diphtheria toxin receptor, LRP-1, or apolipoprotein E were discouraging [52], although earlier studies indicated that these targets were relevant for nanoparticle uptake in BECs [56–58]. Studies using unconjugated antibodies targeting LRP-1 also failed to prove that LRP-1 was a viable target for specific uptake by the brain endothelium [19]. It is feasible that the widespread expression of the aforementioned targets in peripheral vasculature may reduce the extent of liposome availability for uptake at BECs. pared to stealth liposomes conjugated with non-immune IgG [52]. In contrast, liposomes conjugated to proteins putatively capable of targeting BECs, e.g., i) cross-reacting material (CRM) with affinity for an endogenous diphtheria toxin receptor; ii) angiopep-2 with affinity for LRP-1; iii) COG133 with affinity for apolipoprotein E, all failed to exhibit higher uptake compared to non-immune IgG-conjugated liposomes [52]. In particular, the observations made on liposomes targeting the diphtheria toxin receptor, LRP-1, or apolipoprotein E were discouraging [52], although earlier studies indicated that these targets were relevant for nanoparticle uptake in BECs [56–58]. Studies using unconjugated antibodies targeting LRP-1 also failed to prove that LRP-1 was a viable target for specific uptake by the brain endothelium [19]. It is feasible that the widespread expression of the aforementioned targets in peripheral vasculature may reduce the extent of liposome availability for uptake at BECs. The number of antibodies present on the surface of antibody-functionalized gold na-

The uptake of RI7217-conjugated liposomes at the BBB is significantly higher com-

The number of antibodies present on the surface of antibody-functionalized gold nanoparticles and cargo-loaded stealth liposomes influenced the targeting to BECs in vivo. Hence, the highest density out of a selection of different densities (0.15, 0.3, and 0.6 ∗ 103 antibodies/µm2 ) led to the highest binding and uptake [59]. Using gold nanoparticles conjugated with targeting antibodies with different affinity for the transferrin receptor [40], or lowering the avidity inversely led to higher uptake of targeted nanoparticles both in vivo in the adult mouse and in vitro in isolated primary mouse BECs from adult mice [54] (Figure 3). Examining the influence of the avidity of the targeting antibodies, using bispecific antibodies targeting both the transferrin receptor and amyloid beta (i.e., mono-valent binding to the transferrin receptor, this approach resulted in higher binding and uptake when compared to low-affine, bivalent monospecific antibodies both in vivo and in vitro [54]. noparticles and cargo-loaded stealth liposomes influenced the targeting to BECs in vivo. Hence, the highest density out of a selection of different densities (0.15, 0.3, and 0.6 ∗ 103 antibodies/μm2 ) led to the highest binding and uptake [59]. Using gold nanoparticles conjugated with targeting antibodies with different affinity for the transferrin receptor [40], or lowering the avidity inversely led to higher uptake of targeted nanoparticles both in vivo in the adult mouse and in vitro in isolated primary mouse BECs from adult mice [54] (Figure 3). Examining the influence of the avidity of the targeting antibodies, using bispecific antibodies targeting both the transferrin receptor and amyloid beta (i.e., monovalent binding to the transferrin receptor, this approach resulted in higher binding and uptake when compared to low-affine, bivalent monospecific antibodies both in vivo and in vitro [54].

*Pharmaceutics* **2022**, *14*, x FOR PEER REVIEW 7 of 19

**Figure 3.** Uptake in the adult mouse brain of gold-labeled nanoparticles (AuNPs) targeted to the BBB by anti-transferrin receptor antibodies varying in affinity. Identical antibodies were studied prior for BBB transport without conjugation [44]. (**A**) In whole brain homogenates, there is a clear distinction between the different transferrin receptor (TfR)-targeted variants with respect to their accumulation. (**B**) The TfR-targeted AuNPs accumulate in the capillary fraction with 0.2, 0.5, and 1.1%ID/g for anti-TfRA, anti-TfRD, and anti-TfRA/BACE1. (**C**) In fractions containing brain parenchyma, detection of AuNPs indicates transport across the BBB. Accumulation is mainly seen for the low-affinity anti-TfRD compared to the high-affinity anti-TfRA variant AuNPs. Anti-TfRA/BACE1 AuNPs are superior to the other TfR-targeted variant AuNPs with a mean parenchymal accumulation of 0.23% ID/g compared to 0.04 and 0.08% ID/g for anti-TfRA and anti-TfRD, respectively. Modified from [54]. The uptake of nanoparticles may be further enhanced by changing the shape of nanoparticles, provided they are constructed by a relevant material [60,61]. In vitro studies **Figure 3.** Uptake in the adult mouse brain of gold-labeled nanoparticles (AuNPs) targeted to the BBB by anti-transferrin receptor antibodies varying in affinity. Identical antibodies were studied prior for BBB transport without conjugation [44]. (**A**) In whole brain homogenates, there is a clear distinction between the different transferrin receptor (TfR)-targeted variants with respect to their accumulation. (**B**) The TfR-targeted AuNPs accumulate in the capillary fraction with 0.2, 0.5, and 1.1%ID/g for anti-TfRA, anti-TfRD, and anti-TfRA/BACE1. (**C**) In fractions containing brain parenchyma, detection of AuNPs indicates transport across the BBB. Accumulation is mainly seen for the low-affinity anti-TfRD compared to the high-affinity anti-TfRA variant AuNPs. Anti-TfRA/BACE1 AuNPs are superior to the other TfR-targeted variant AuNPs with a mean parenchymal accumulation of 0.23% ID/g compared to 0.04 and 0.08% ID/g for anti-TfRA and anti-TfRD, respectively. Modified from [54]. Data are presented as mean ± SEM (n = 7–8, Kruskal-Wallis test with Dunn's multiple comparisons post hoc test) with \* *p* < 0.05 and \*\*\* *p* < 0.001. %ID/g: percentage of injected dose per gram.

showed that rod-shaped polymeric nanoparticles targeted to the transferrin receptor underwent seven-fold higher uptake compared to spherical particles [60], clearly warranting further pursuit in vivo. The nanoparticles' zeta potential is also important with slightly The uptake of nanoparticles may be further enhanced by changing the shape of nanoparticles, provided they are constructed by a relevant material [60,61]. In vitro studies showed that rod-shaped polymeric nanoparticles targeted to the transferrin receptor underwent seven-fold higher uptake compared to spherical particles [60], clearly war-

ranting further pursuit in vivo. The nanoparticles' zeta potential is also important with slightly negative potential being optimal for uptake [11–13]. When present in blood plasma, nanoparticles tend to absorb blood-circulating proteins forming a so-called protein corona so significant that it may sterically block for binding of the targeting protein to its receptor [61]. Previous research on the liposomal protein corona may have suffered from significant methodological limitations making this issue too problematic as justified in more controlled experiments, showing that contaminating proteins may have interfered with the analysis of the protein corona [62–64]. To prevent a potential unwanted influence of protein corona on the targeting potential of the nanoparticles, the nanoparticles can be conjugated to their targeting antibody bridged in-between by PEG molecules, which simultaneously will limit their likelihood of being taken up in the reticuloendothelial system (RES) [9,10].

The possibility of using the transferrin receptor for targeting nanoparticles to the BECs has also been pursued in studies using dual targeting approaches in which transferrin is linked to the surface of nanoparticles in conjunction with other peptides. Studies were mainly performed in vitro, with additional biodistribution studies in vivo using nanoparticles conjugated with transferrin and cell-penetrating peptides (CPPs), or rabies virus glycoprotein (RVG) [65–67]. Although these approaches bear great potential, they warrant direct comparisons with antibodies targeting the transferrin receptor for efficient drug delivery to the brain. Other strategies examined the transferrin receptor targeting peptide T7 (aka T7-LS) bound to the surface of liposomes containing the chemotherapeutic vincristine and reported a significantly higher pharmacological effect compared to the targeting of liposomes bound to other transferrin receptor targeting peptides B6 and T12 [68].

Despite being available for experimental use for more than three decades, the transferrin receptor is by far still considered the most relevant target for nanoparticle drug delivery. It is only a few other candidates, such as CD98, GLUT1, basigin (CD147), and the insulin receptor that were being taken into consideration as an alternative, but other than the insulin receptor, these receptors remain unexploited for their targetability to BECs with regard to nanoparticles [19].

#### **6. Anti-Transferrin Receptor Antibodies Weakened in Affinity or Lowered in Avidity Facilitate Nanoparticle Transport through BECs**

Anti-transferrin receptor-targeted monoclonal antibodies weakened in affinity for the receptor readily undergo transport across the BBB in a dose-dependent manner, becoming detectable in neurons [40]. Bispecific antibodies, with one domain targeting the transferrin receptor at high affinity and the other domain directed towards a putative therapeutic target relevant for treating Alzheimer's disease, i.e., beta-secretase 1, lead to reduction of amyloidogenic peptide formation in the brain [41]. Unfortunately, studies have not yet been performed to examine the efficacy of targeted nanoparticles with respect to repeated or chronic dosing regimens. Such evaluations would help to understand the amounts of therapeutics encapsulated within nanoparticles that can be accumulated inside the brain.

Antibodies with low affinity for the transferrin receptor seem to follow a cellular route identical to that of high-affine antibodies, as prior exposure to high-affine antibodies leads to reduced transport of low-affine antibodies across the BBB both in vivo and in vitro [69]. The two different antibodies differ in that antibodies with low affinity are not directed towards lysosomes to the same extent as antibodies with high affinity [69]. Further information on intracellular transport relying on anti-transferrin receptor antibodies comes from the study on the subcellular distribution of anti-transferrin receptor antibody-conjugated gold nanoparticles [54] (Figure 4). This revealed gold particles in BECs, and when conjugated with low-affine or low-avidity antibodies, the gold particles were also detected in neurons, further arguing for transport across BECs. The targeted gold nanoparticles were apparent in BECs in clearly identifiable vesicular structures, which might represent sorting endosomes and lysosomes. The study did not identify gold nanoparticles fusing with the abluminal membrane, but this does not exclude the transcellular transport of the nanoparticles through the BBB. This uncertainty comes from the observation of a low number of sequestered

particles, and that the electron microscopy data were collected from thin sections, typically less than 100 nm. In turn, isolated mouse BECs arranged in hanging cell culture inserts with defined BBB properties revealed transcellular transport of gold nanoparticles when conjugated with low-affine or low-avidity antibodies, which supports the observation of nanoparticle transport across the BBB. Notably, the electron microscopy studies did not show signs of obstructive accumulation of the gold nanoparticles sized approximately 75 nm near the basement membrane, which may be a major restraint for nanoparticle trafficking in the brain's extracellular volume after release at the abluminal side of the BECs [9]. cellular transport of the nanoparticles through the BBB. This uncertainty comes from the observation of a low number of sequestered particles, and that the electron microscopy data were collected from thin sections, typically less than 100 nm. In turn, isolated mouse BECs arranged in hanging cell culture inserts with defined BBB properties revealed transcellular transport of gold nanoparticles when conjugated with low-affine or low-avidity antibodies, which supports the observation of nanoparticle transport across the BBB. Notably, the electron microscopy studies did not show signs of obstructive accumulation of the gold nanoparticles sized approximately 75 nm near the basement membrane, which may be a major restraint for nanoparticle trafficking in the brain's extracellular volume after release at the abluminal side of the BECs [9].

nanoparticles fusing with the abluminal membrane, but this does not exclude the trans-

*Pharmaceutics* **2022**, *14*, x FOR PEER REVIEW 9 of 19

**Figure 4.** Detection of anti-transferrin receptor IgG conjugated gold-labeled nanoparticles (AuNPs) using transmission electron microscopy (TEM) in a normal adult mouse brain. Anti-transferrin receptor antibodies vary in affinity. (**A**–**B**) AuNPs are not detected in brain capillaries of mice in the mPEG (no IgG added) or isotype (non-immune) IgG groups. (**C**–**E**) In contrast, the AuNPs targeted with anti-transferrin receptor IgG are found in BECs (arrows). The AuNPs are detected in BECs confined to vesicular structures, suggesting receptor-mediated endocytosis as the uptake mechanism. (**F**–**G**) In brain parenchyma, AuNPs are not detected in mice in the mPEG or isotype (non-immune) IgG groups. (**H**–**J**) AuNPs are seen in brain parenchyma of mice treated with all transferrin receptor (TfR)-targeted variants, among which they are most easily detected in the anti-TfRA/BACE1 group (**J**). The sites for transport of the AuNPs may derive from transport across either BECs or post-capillary venules (see text body). All AuNPs detected in the brain parenchyma were analyzed using energy-dispersive X-ray spectroscopy (EDS) to validate the true presence of gold in the electron-dense points (**K**–**M**).  Modified from [54]. **Figure 4.** Detection of anti-transferrin receptor IgG conjugated gold-labeled nanoparticles (AuNPs) using transmission electron microscopy (TEM) in a normal adult mouse brain. Anti-transferrin receptor antibodies vary in affinity. (**A**,**B**) AuNPs are not detected in brain capillaries of mice in the mPEG (no IgG added) or isotype (non-immune) IgG groups. (**C**–**E**) In contrast, the AuNPs targeted with anti-transferrin receptor IgG are found in BECs (arrows). The AuNPs are detected in BECs confined to vesicular structures, suggesting receptor-mediated endocytosis as the uptake mechanism. (**F**,**G**) In brain parenchyma, AuNPs are not detected in mice in the mPEG or isotype (non-immune) IgG groups. (**H**–**J**) AuNPs are seen in brain parenchyma of mice treated with all transferrin receptor (TfR)-targeted variants, among which they are most easily detected in the anti-TfRA/BACE1 group (**J**). The sites for transport of the AuNPs may derive from transport across either BECs or post-capillary venules (see text body). All AuNPs detected in the brain parenchyma were analyzed using energy-dispersive X-ray spectroscopy (EDS) to validate the true presence of gold in the electron-dense points (**K**–**M**).  Modified from [54].

#### **7. A Mechanistic Approach to an Understanding of Trafficking of Transferrin Receptor-Targeting Liposomes Based on Studies of Iron-Transferrin and Unconjugated Anti-Transferrin Receptor Antibody Trafficking in BECs**

The paucity of studies examining intracellular trafficking of nanoparticles in BECs in vivo limits the available information about their transport mechanisms. Some lessons may be learned from comparison of endogenous transferrin and unconjugated, targeted anti-transferrin receptor antibodies. Although this difference obviously should be taken into account, the low-affinity antibodies and low-affinity antibodies conjugated to PEGylated liposomes share great similarities in transport through the BBB with unconjugated lowaffinity and low-affinity anti-transferrin receptor antibodies [40,54].

#### *7.1. Blood to Endothelium Transport*

Endogenous transferrin enters the BECs after the interaction with the transferrin receptor on the luminal surface, facilitating subsequent formation of clathrin-coated pits, and eventually, formation of endosomes [9,16,24]. The endosomes have a slightly acidic pH, which promotes detaching iron from transferrin [9,16]. Consequently, the unbound iron can cross the endosomal membrane via divalent metal transporter 1 (DMT1), which makes the iron available in the cytosol [9,25]. In parallel, the iron-depleted apo-transferrin residing inside the endosome loses its affinity for the transferrin receptor, and is thought to undergo retro-endocytosis to the luminal surface of the endothelial cell [9,16].

#### *7.2. Endothelium to Brain Transport*

Whereas the docking and endosomal formation relate to the affinity of the transferrin receptor, it can be argued that the intracellular trafficking of the endosomes follow routes that occur independently of the luminal receptor internalization [9,16,51]. BECs contain RAB4 and RAB7 proteins specific for early and later sorting endosomes [70,71]. The abluminal membrane of the BECs also contains the protein TSG101 (tumor susceptibility gene 101), which takes part in exocytosis. This suggests that BECs contain organelles fully capable of handling transferrin receptor-containing vesicles that present themselves initially as early forming endosomes with the capacity to fuse with late endosomes, eventually leading to fusion with the abluminal membrane and exocytosis [72]. Therefore, the process of sorting transferrin receptor-containing vesicles is likely to be in two ways: Both morphological and pharmacological studies favor receptor-mediated endocytosis taking place at the luminal side leading to formation of early endosomes. This is followed by a vaguely understood trafficking of late endosomes directed towards fusion with the abluminal surface [9,16].

A morphological approach to detect transferrin at the ultrastructural level in rats subjected to the brain in situ perfusion failed to detect HRP-conjugated iron-transferrin near the abluminal side, which, in turn, was outnumbered by the presence of HRP-transferrin in multiple vesicular-like structures near the luminal side of the BECs [58]. However, as mentioned earlier, in the developing brain BECs are enriched in vesicles involved in transcellular trafficking. This, together with the observation that the developing brains have relatively higher expression of transferrin receptors [30,31], could account for directed transcellular trafficking of iron-transferrin-containing vesicles through the BECs. Interestingly, intracarotid perfusion with OX26-conjugated colloidal gold enabled detection at the abluminal side of the brain endothelium [58]. Although this study did not quantify the transport of the gold-labeled OX26, the appearance near the abluminal side may represent transferrin receptor-containing vesicles available for fusion at the abluminal side. A conclusion may be that as only a minor fraction of iron-transferrin within the transferrincontaining vesicles moves towards the abluminal side of the BEC, only a minor fraction of such vesicles including their content are released at the abluminal side of the BECs.

The binding to high-affine anti-transferrin receptor antibodies also leads to the formation of endocytotic vesicles that mainly localize near the luminal membrane [9,16]. The uptake and transport of high-affine, anti-transferrin receptor antibodies within the BECs are likely to follow the same route as that of iron-transferrin. However, differences may

occur, as the binding of the high-affine antibody to the transferrin receptor is thought not to be reversible, leading to accumulation of the antibody within the endosomal-lysosomal compartment [16]. Noteworthy, later studies addressing the fate of the anti-transferrin receptor antibodies showed that the complexes are incorporated in lysosomes rather than being directed towards release at the abluminal membrane [69]. A study in a mouse model with human transferrin receptors revealed that high-affine anti-transferrin receptor antibodies could undergo transport across the BBB [43]. However, it is very difficult to compare the consequences of binding affinity of anti-transferrin receptor antibodies between species concerning the capability to undergo transport at the BBB. The mechanisms that enable high-affine and low-affine antibodies to detach from the transferrin receptor within the endosome is poorly understood, but possibly, the acidic environment in the endosome facilitates the detachment of antibodies from the receptor. Even if a minor fraction of antibodies bound to the transferrin receptor is released in the acidic endosomal environment, a fraction of the high-affinity antibody would be released and would move further into the brain's extracellular space. *Pharmaceutics* **2022**, *14*, x FOR PEER REVIEW 11 of 19 uptake and transport of high-affine, anti-transferrin receptor antibodies within the BECs are likely to follow the same route as that of iron-transferrin. However, differences may occur, as the binding of the high-affine antibody to the transferrin receptor is thought not to be reversible, leading to accumulation of the antibody within the endosomal-lysosomal compartment [16]. Noteworthy, later studies addressing the fate of the anti-transferrin receptor antibodies showed that the complexes are incorporated in lysosomes rather than being directed towards release at the abluminal membrane [69]. A study in a mouse model with human transferrin receptors revealed that high-affine anti-transferrin receptor antibodies could undergo transport across the BBB [43]. However, it is very difficult to compare the consequences of binding affinity of anti-transferrin receptor antibodies between species concerning the capability to undergo transport at the BBB. The mechanisms that enable high-affine and low-affine antibodies to detach from the transferrin receptor within the endosome is poorly understood, but possibly, the acidic environment in the endosome facilitates the detachment of antibodies from the receptor. Even if a minor fraction of antibodies bound to the transferrin receptor is released in the acidic endosomal environ-

It goes beyond doubt that the optimal transport of targeted nanoparticles at the BBB depends on the binding of anti-transferrin receptor antibodies to the endothelial surface [68]. The transport of nanoparticles through BECs appears to depend on the affinity or low avidity of the anti-transferrin receptor antibody. Nanoparticles, therefore, may undergo transport through BECs similar to therapeutically active molecules like enzymes or other proteins directly conjugated to anti-transferrin receptor antibodies [73] (Figure 5). ment, a fraction of the high-affinity antibody would be released and would move further into the brain's extracellular space. It goes beyond doubt that the optimal transport of targeted nanoparticles at the BBB depends on the binding of anti-transferrin receptor antibodies to the endothelial surface [68]. The transport of nanoparticles through BECs appears to depend on the affinity or low avidity of the anti-transferrin receptor antibody. Nanoparticles, therefore, may undergo transport through BECs similar to therapeutically active molecules like enzymes or other proteins directly conjugated to anti-transferrin receptor antibodies [73] (Figure 5).

**Figure 5.** Working model of transcytosis-mediated nanoparticle delivery to the brain. Recent study provides evidence that high-affine anti-transferrin receptor IgG conjugated to liposomes mainly undergo transport into the brain at the site of post-capillary venules [14]. This observation counteracts that of unconjugated antibodies that passes the BBB at the site of brain capillaries when designed to be low in affinity or avidity [42]. Targeting the latter antibodies to gold nanoparticles leads to **Figure 5.** Working model of transcytosis-mediated nanoparticle delivery to the brain. Recent study provides evidence that high-affine anti-transferrin receptor IgG conjugated to liposomes mainly undergo transport into the brain at the site of post-capillary venules [14]. This observation counteracts that of unconjugated antibodies that passes the BBB at the site of brain capillaries when designed to be low in affinity or avidity [42]. Targeting the latter antibodies to gold nanoparticles leads to capillary transport in vitro using isolated brain capillaries [58] suggesting that accumulation of targeted nanoparticles in brain parenchyma in vivo may occur via transport across capillaries as well. Studies concerning transport into brain across post-capillary venules using transferrin receptortargeted low-affine antibodies (lower right) have not been performed but can be stipulated to lead to enhanced transport compared to the use of corresponding antibodies with high affinity. Red bullets: high-affine anti-transferrin receptor IgG conjugated liposomes. Yellow bullets: low-affine or low-avidity anti-transferrin receptor IgG conjugated to liposomes. Drawing created with BioRender, inspired and modified from [14].

#### **8. Post-Capillary Venules Denote an Alternate Route for Transport**

The use of 2-photon microscopy (2PM) for in vivo studies has revolutionized the concept of how the brain works and has recently entered the field of the BBB research [74]. 2 PM visualizes fluorescently labeled molecules in the brain with a sub-micron spatial resolution and allows for visualization of individual nanoparticles in blood vessels to a depth of 600 µm below the pial surface in vivo [74]. Recent studies using 2PM on the transferrin receptor-targeted liposomes demonstrated how endothelial cells of both brain capillaries and post-capillary venules in contrast to arterioles handle transferrin receptortargeted liposomes at the level of a single nanoparticle [14] (Figure 6). The major finding was that the liposomes targeted with high-affine RI7217 were released to the brain almost exclusively from endothelial cells of the post-capillary venules, with negligible results. *Pharmaceutics* **2022**, *14*, x FOR PEER REVIEW 13 of 19

**Figure 6.** Example of in vivo 2PM data, showing three-dimensional reconstruction of cortical microvasculature (left), and the distribution of intravenously-injected transferrin receptor-targeted liposomes residing at the BBB interface (right). The transferrin receptor-targeted liposomes associate primarily to capillaries, then venules, but are absent in arterioles. The images were collected 2 h post injection. Modified from [14]. **Figure 6.** Example of in vivo 2PM data, showing three-dimensional reconstruction of cortical microvasculature (**left**), and the distribution of intravenously-injected transferrin receptor-targeted liposomes residing at the BBB interface (**right**). The transferrin receptor-targeted liposomes associate primarily to capillaries, then venules, but are absent in arterioles. The images were collected 2 h post injection. Modified from [14].

**9. Targeting Nanoparticles to the Brain Endothelium in Pathological Conditions** The use of targeting approaches to promote enhanced drug delivery to the brain in pathology is only coming of age. Targeting approaches in conditions with cerebral pathology can roughly be separated into attempts to treat the brain in acute and chronic conditions. In acute conditions, e.g., ischemic stroke and traumatic brain injury, liposomes are advantageous among nanoparticles because they can be formulated to contain degradable lipids with enzymes like matrix metalloproteinases and phospholipases known to be increased in expression and released from the brain in acute pathology [76–79]. In terms of delivery to the brain, the liposomes will easily enter the affected brain regions as the vasculature is deteriorating, leading to the opening of the BBB. The liposomes are expected to start to degrade once they enter areas of the brain with a raised expression of, e.g., matrix metalloproteinases. This approach does not demand the liposomes to be functionalized by conjugation to a targeting antibody, but the targeting approach could allow for One study showed the contribution of endothelium at capillaries [14], which is scientifically provocative, considering previous investigations using this antibody to study transport across the brain endothelium. The release of the liposomes from the post-capillary venules is surprising, and the mechanisms and explanation for this observation will need further study. The barrier formed by the endothelial cells of the post-capillary venules is less tight at the venous side with respect to the number of tight junctions compared to BECs [75]. This could principally allow nanoparticles to enter the brain via paracellular transport, but counteracting this notion, the targeted nanoparticles were clearly taken up by the endothelial cells of the post-capillary venules before entering the brain on the abluminal side, verifying transendothelial transport of the targeted liposomes. In all, the density of transferrin receptor-targeted nanoparticles, being highest in capillaries, did not translate to efficient transport of nanoparticles to the brain. Thus, the BBB is highly heterogeneous regarding transport mechanisms, and, in particular, the ability to transcytose large constructs across the BBB [14].

more widespread uptake of the liposomes in the surrounding areas of a central pathology, e.g., in ischemic stroke, the penumbra zone, where the BBB often remains intact [80]. Targeting to the BECs could enhance the liposomal delivery, which might allow, e.g., en-The study of Kucharz et al. [14] clearly opens for novel considerations on the transport of targeted nanoparticles to the brain. An interesting consideration related to prior studies

hanced pharmacological preconditioning using focused ultrasound and microbubble

served, e.g., post stroke [82,83]. As such, the obstructed blood flow may limit the ability of the blood circulation to deliver the liposomes to the relevant, damaged areas in the

Recent efforts aim to utilize a dual targeting approach using antibodies targeting the transferrin receptor and intercellular adhesion molecule-1 (ICAM1), both conjugated to liposomes. This resulted in higher binding compared to liposomes targeted to only transferrin receptor or ICAM1 antibodies alone [84]. This approach led to enhanced delivery of

brain.

demonstrating targeted liposomes within the brain [11–13,54] is that they could also have passed into the brain via this hitherto overseen route at post-capillary venules.

#### **9. Targeting Nanoparticles to the Brain Endothelium in Pathological Conditions**

The use of targeting approaches to promote enhanced drug delivery to the brain in pathology is only coming of age. Targeting approaches in conditions with cerebral pathology can roughly be separated into attempts to treat the brain in acute and chronic conditions. In acute conditions, e.g., ischemic stroke and traumatic brain injury, liposomes are advantageous among nanoparticles because they can be formulated to contain degradable lipids with enzymes like matrix metalloproteinases and phospholipases known to be increased in expression and released from the brain in acute pathology [76–79]. In terms of delivery to the brain, the liposomes will easily enter the affected brain regions as the vasculature is deteriorating, leading to the opening of the BBB. The liposomes are expected to start to degrade once they enter areas of the brain with a raised expression of, e.g., matrix metalloproteinases. This approach does not demand the liposomes to be functionalized by conjugation to a targeting antibody, but the targeting approach could allow for more widespread uptake of the liposomes in the surrounding areas of a central pathology, e.g., in ischemic stroke, the penumbra zone, where the BBB often remains intact [80]. Targeting to the BECs could enhance the liposomal delivery, which might allow, e.g., enhanced pharmacological preconditioning using focused ultrasound and microbubble treatment [81]. However, it should be noted that many acute conditions are also associated with obstructed blood flow due to pericyte-mediated constriction of capillaries, as observed, e.g., post stroke [82,83]. As such, the obstructed blood flow may limit the ability of the blood circulation to deliver the liposomes to the relevant, damaged areas in the brain.

Recent efforts aim to utilize a dual targeting approach using antibodies targeting the transferrin receptor and intercellular adhesion molecule-1 (ICAM1), both conjugated to liposomes. This resulted in higher binding compared to liposomes targeted to only transferrin receptor or ICAM1 antibodies alone [84]. This approach led to enhanced delivery of liposomes encapsulating the anti-inflammatory cytokine TNF-alpha in a model of acute brain inflammation [84].

In chronic brain disorders, like Alzheimer's disease and Parkinson's disease, the brain does not increase the expression of liposome-degradable enzymes like metalloproteinases and phospholipases to the same extent as seen in acute neuropathology, and therefore, the strategy of substrate degradable liposomes may not apply. Instead, it may be advantageous that the BEC expression of the transferrin receptor remains unchanged in Alzheimer's disease [85], which further justifies attempts to target transferrin receptors expressed by BECs in the neurodegenerative brain expected to have a near intact BBB.

#### **10. Conclusions**

BECs are practically incapable of uptake of native liposomes from the blood, which requires the addition of targeted molecules. Experience gained from different targeting approaches justifies the choice of antibodies targeting the transferrin receptor for targeted delivery of nanoparticles to BECs. Liposomes functionalized by conjugated anti-transferrin receptor antibodies are taken up by endothelial cells at both brain capillaries and postcapillary venules. Modulating the number of transferrin receptor-targeted antibodies shows that limiting the number of targeting antibodies conjugated to liposomes reduces uptake in BECs. In comparison, lowering the affinity of the targeting antibodies or implementing bispecific antibodies with low avidity increases transport by BECs into the brain parenchyma. With increasing evidence of successful preclinical trials and advances in biochemical and analytical approaches, the transferrin receptor-targeted nanoparticles have great promise for future use in drug delivery as they evidently pass the BBB.

**Author Contributions:** Conceptualization, M.S.T. and T.M.; Writing—original draft preparation, M.S.T. and T.M.; Writing—review and editing, K.B.J., K.K. and M.L. All authors have read and agreed to the published version of the manuscript.

**Funding:** Parts of the data by the authors presented in this manuscript were obtained by generous support from the Lundbeck foundation and by a grant from Agnes og Poul Friis Fond to Martin Lauritzen and Krzysztof Kucharz.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** None.

**Conflicts of Interest:** The authors have no conflict of interest to declare.

#### **References**

## *Article* **pH-Responsive Lipid Nanoparticles Achieve Efficient mRNA Transfection in Brain Capillary Endothelial Cells**

**Yu Sakurai 1,\* ,† , Himeka Watanabe 1,†, Kazuma Nishio 1,†, Kohei Hashimoto 1,†, Atsuki Harada 1,† , Masaki Gomi 2 , Masayoshi Suzuki 1 , Ryotaro Oyama 2 , Takumi Handa 1 , Risa Sato 1 , Hina Takeuchi 1 , Ryoga Taira 1 , Kenta Tezuka 1 , Kota Tange 3 , Yuta Nakai 3 , Hidetaka Akita 1,\* and Yasuo Uchida 1,\***

**Abstract:** The blood–brain barrier (BBB), which is comprised of brain capillary endothelial cells, plays a pivotal role in the transport of drugs from the blood to the brain. Therefore, an analysis of proteins in the endothelial cells, such as transporters and tight junction proteins, which contribute to BBB function, is important for the development of therapeutics for the treatment of brain diseases. However, gene transfection into the vascular endothelial cells of the BBB is fraught with difficulties, even in vitro. We report herein on the development of lipid nanoparticles (LNPs), in which mRNA is encapsulated in a nano-sized capsule composed of a pH-activated and reductive environmentresponsive lipid-like material (ssPalm). We evaluated the efficiency of mRNA delivery into nonpolarized human brain capillary endothelial cells, hCMEC/D3 cells. The ssPalm LNPs permitted marker genes (GFP) to be transferred into nearly 100% of the cells, with low toxicity in higher concentration. A proteomic analysis indicated that the ssPalm-LNP had less effect on global cell signaling pathways than a Lipofectamine MessengerMAX/GFP-encoding mRNA complex (LFN), a commercially available transfection reagent, even at higher mRNA concentrations.

**Keywords:** lipid nanoparticle; ssPalm; mRNA transfection; blood–brain barrier; hCMEC/D3 cells; cell toxicity; SWATH-MS; translation; chaperonin-containing TCP-1

#### **1. Introduction**

Various cells are currently available as in vitro models for the BBB model. However, none of them completely reflect in vivo brain capillary endothelial cells.  Another example, human-induced pluripotent stem cell-derived brain microvascular endothelial cells (hiPS-BMECs), show significantly lower expression levels of the multidrug resistance protein (MDR1/P-gp), and therefore, they lack drug efflux activity [2]. Therefore, the transfection of genes that can complement the protein to the

**Citation:** Sakurai, Y.; Watanabe, H.; Nishio, K.; Hashimoto, K.; Harada, A.; Gomi, M.; Suzuki, M.; Oyama, R.; Handa, T.; Sato, R.; et al. pH-Responsive Lipid Nanoparticles Achieve Efficient mRNA Transfection in Brain Capillary Endothelial Cells. *Pharmaceutics* **2022**, *14*, 1560. https://doi.org/10.3390/ pharmaceutics14081560

Academic Editors: William M. Pardridge, Patrick T. Ronaldson and Inge S. Zuhorn

Received: 23 May 2022 Accepted: 24 July 2022 Published: 27 July 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

level of that in the in vitro model cells would be a powerful tool for establishing a more valid BBB model that can mimic in vivo. However, the transfection of brain capillary endothelial cells has been reported to be difficult in some studies. It has been reported that, in primary cerebral vascular endothelial cells, less than 5% of the cells are transfected with plasmid DNA when a commercially available transfection reagent, LyoVec is used [3]. Such a low transfection efficiency has also been reported for hCMEC/D3 cells. For example, when hCMEC/D3 cells were transduced with a P-gp fusion gene and the green fluorescent protein (GFP) using the Lentivirus vector, the transfection efficiency was only approximately 10% [4]. One of the reasons for the low transfection efficiency is attributed to poor cellular uptake. In fact, it has been reported that the percentage of cells that took up double-stranded DNA as an agonist of the toll-like receptor was less than half that of hepatocytes and macrophages using a cationic polymer [5]. While there is no doubt that hCMEC/D3 cells are potentially useful as an in vitro BBB model, a reliable method for introducing specific genes remains to be developed. Lipofectamine is widely used, but a major drawback to its use is that the cationic liposomes contained in it produce unintended cytotoxicity. In human endothelium-derived cells (HUVECs), lipofectamine 2000 has been reported to inhibit cell proliferation, reduce the expression of various proteins, and cause an unfolded protein response (UPR) [6]. We here hypothesize that the use of an uncharged neutral nanoparticle would be more feasible for use in conjunction with endothelial cells.

To achieve a more efficient gene delivery into human brain capillary endothelial cells, hCMEC/D3, we developed lipid nanoparticles (LNPs) that contain encapsulated mRNA [7]. This LNP was designed to satisfy two fundamental properties, namely, high biocompatibility and high intracellular auto-degradability. To achieve this, we developed an ss-cleavable pH-activated lipid-like material (ssPalm), which was equipped with tertiary amines that develops a positive charge in response to the acidic pH and a disulfide-bonded unit that undergoes molecular disintegration in the intracellular reductive environment (Figure S1). When the ssPalm molecules are reconstituted into the LNPs (ssPalm-LNPs), the entire LNP had an apparent acid dissociation constant (p*Ka*) of around 6.3. Therefore, under physiological conditions, the ssPalm-LNP would behave as a neutral nanoparticle. However, in the acidic environment of endosomes, they would become positively charged and then fuse with the negatively charged cell membrane. The fusion between LNP and cell membrane in acidic endosome led to membrane disruption and subsequent endosomal escape of LNP to cytosol. The ssPalm also reacts with reducing agents such as the glutathione present in the cell and disintegrates, thus releasing the loaded nucleic acid into the cytoplasm [7]. Controlling the intracellular dynamics of encapsulated nucleic acids through such multi-step actions of these functional units enabled mRNA to be efficiently introduced into cells that are generally considered to be relatively resistant to transfection [8]. In this study, we attempted to apply the ssPalm-LNP for the transfection of mRNA into hCMEC/D3 cells, for which introducing genes has been difficult.

Regarding the evaluation of adverse effects after the transfection, a comprehensive analysis of the protein expression level would be useful. The SWATH-MS (sequential window acquisition of all theoretical fragment ion spectra mass spectrometry) method is one of the recently developed quantitative proteomics methods, whose quantitative accuracy is higher than previous comprehensive proteomics technology [9]. Using this methodology, we compared the variation in the level of protein expression after the transfection with the ssPalm-LNP.

Based on these analyses, we report herein on the advantage of using the ssPalm-LNP in terms of achieving a high mRNA transfection to the non-polarized human brain capillary cells, hCMEC/D3, with low adverse effects.

#### **2. Materials and Methods**

#### *2.1. Cell Culture*

The detail in the hCMEC/D3 culture procedure was almost the same as previously reported but slightly modified [1,10,11]. The hCMEC/D3 cells were cultured in Endo-GRO complete Media Kit (Merck-Millipore, Burlington, MA, USA) on the plate coated with Cultrex Rat type-I collagen (R&D Systems, Minneapolis, MN, USA) at 37 ◦C under a 5% CO2 atmosphere for 3–4 days, and maintained until used for mRNA transfection.

#### *2.2. Preparation of ssPalm-LNP Encapsulating GFP-Encoding mRNA*

The GFP-encoding mRNA modified with 5-methoxyuridine was obtained from TriLink Biotech (San Diego, CA, USA). The ssPalm-LNP using SS-OP (provided by NOF COR-PORATION), 1,2-dioleoyol-*sn*-glycerophosphocholine (DOPC, NOF CORPORATION), cholesterol (Chol, Sigma-Aldrich, Burlington, MA, USA) and poly(ethylene) glycol (average molecular weight 2000)-1,2-dimyrisotyl-*sn*-gycerol (PEG-DMG, NOF CORPORA-TION) was prepared as previously [12,13]. The structures of these lipids are depicted in Figure S1. We formulated mRNA into LNP using ethanol dilution methods, in which lipid molecules/mRNA was spontaneously assembled by gradual decrease in ethanol concentration, based on previously published methodology [14]. Briefly, 3 µg of GFP-encoding mRNA, dissolved in 45 µL of 25 mM malic buffer (30 mM NaCl, pH 3.0), was gradually added to the lipid mixture (total 131.5 nmol (SS-OP/DOPC/Chol 52.5/7.5/35.0) with 3.95 nmol PEG-DMG) in 30.26 µL of ethanol. The solution was then further diluted with 1 mL of 20 mM 2-(N-morpholino)ethanesulfonic acid) (MES) buffer (pH 5.5, 30 mM NaCl) under vigorous mixing with a vortex mixer. The resulting mixture was diluted with 3 mL of MES buffer and then ultrafiltered with an Amicon Ultra-4 (Merck-Millipore, molecular weight cut off: 100 kDa). The concentrate was again ultrafiltered after the dilution with phosphate-buffered saline without Mg2+ and Ca2+ (PBS). The obtained LNP was characterized using a ZetaSizer Pro (Malvern Panalytical, Malvern, UK). The recovery rate and the encapsulation efficiency of the mRNA were determined by a RiboGreen assay, as previously reported [12,13]. To label the LNP with near-infrared fluorescence, 1,10 -dioctadecyl-3,3,30 ,30 tetramethylindodicarbocyanine (DiD) was added to the lipid mixture at 0.5 mol% to total lipid moles before mixing it with the mRNA solution.

#### *2.3. Preparation of Lipofectamine/GFP-Encoding mRNA Complex (LFN)*

Lipofectamine MessengerMAX (ThermoFisher Scientific, Waltham, CA, USA) was used as a control. According to the manufacturers' protocol, 1.5 µL of Lipofectamine Messenger MAX was incubated with 1000 ng of GFP-encoding mRNA for 10 min.

#### *2.4. Flow Cytometry Analysis of Cellular Uptake and Gene Expression*

hCMEC/D3 cells were plated on a 12-well plate at a density of 7.5 × 104 cells/well 24 h before the addition of the ssPalm LNP and LFN. The cells were incubated with the DiD-labeled ssPalm-LNP at a dose of 300 ng mRNA in 750 µL of culture medium with 5% fetal bovine serum (0.4 µg/mL) for 2 h (cellular uptake) at 37 ◦C. To prepare the DiD-labeled LFN, DiD was first mixed with the Lipofectamine MessengerMAX solution so that the final fluorescence intensity in the LFN solution was the same as that for the ssPalm-LNP solution.

To measure GFP expression, the cells were exposed to the ssPalm-LNP solution that had been diluted with a culture medium at an mRNA concentration of 300 ng in 750 µL (0.4 µg/mL) for 16 h. The dilution volume of the ssPalm-LNP was adjusted based on the mRNA recovery ratio measured by the RiboGreen assay result in each experiment. As a control, the LFN was then added to cells cultured in a 12-well plate and incubated for 16 h. The incubated cells were then washed twice with 1.0 mL of PBS and then trypsinized. The obtained cells were suspended in PBS with 0.5% bovine serum albumin and 0.1% sodium azide and analyzed using a NovoCyte (Agilent Technology, Santa Clara, CA, USA).

#### *2.5. Observation of Cellular Uptake and Gene Expression with Con-Focal Laser Scanning Microscopy*

hCMEC/D3 cells were plated onto a glass-based 8-well chamber plate, which had been pre-coated with type-I collagen, at a density of 500 cells/well 48 h before the addition of

the ssPalm-LNP or LFN. The cells were incubated with the ssPalm-LNP at a concentration of 300 ng/200 µL (0.4 µg/mL) for 48 h at 37 ◦C in the presence of 5% fetal bovine serum. As a control treatment, cells were exposed to LFN at the same dose as the ssPalm LNP. Both the ssPalm-LNP and the LFN complex were first diluted in 200 µL of culture medium. Nuclei were stained by a 10-min incubation in 1.0 µg/mL Hoechst33342. After washing with 200 µL of PBS, cells were observed by a Nikon C1 confocal laser scanning microscope system (Nikon, Tokyo, Japan).

#### *2.6. Evaluation of Cytotoxicity in mRNA Transfection Using ssPalm-LNP and LFN in hCMEC/D3 Cells*

The hCMEC/D3 cells were treated with the ssPalm-LNP containing GFP-encoding mRNA, and the LFN with 1250, 2500, and 6250 ng of GFP-mRNA in 1 mL (1.25, 2.5, and 6.25 µg/mL) for 48 h at 37 ◦C in 5% CO2 in the presence of 5% fetal bovine serum, and observed under a microscope.

#### *2.7. SWATH-MS Analysis in hCMEC/D3 Cells Treated with ssPalm LNP and LFN*

The ssPalm-LNP and the LFN were treated with hCMEC/D3 cells at a concentration of 2.5 µg/mL for 48 h at 37 ◦C in 5% CO2. The SWATH-MS analysis was then performed for the whole cell lysate of hCMEC/D3 cells, as previously described [15,16]. Briefly, after a 48-h treatment, 6 well plates of cells were placed on ice, and the cell surface was immediately washed with ice-cold PBS. A denaturing buffer (7 M guanidium hydrochloride, 0.5 M Tris–HCl (pH 8.5), 10 mM EDTA) was directly added to the cell surface in order to prepare a whole cell lysate. The dissolved cells were subjected to up-and-down strokes in a 27G × 1/2 syringe (Terumo, Tokyo, Japan) to completely lyse the cells. The protein concentration of the whole cell lysate was determined by a BCA assay (Thermo Fisher Scientific Inc., Waltham, MA, USA). A 50 µg sample of protein in the whole cell lysate was reduced, S-carboxymethylated, and purified by methanol-chloroform precipitation. The precipitate was solubilized in a urea buffer containing 0.05% ProteaseMax surfactant (Promega, Madison, WI, USA), and the proteins were digested with lysyl endopeptidase (Lys-C, Wako Pure Chemical Industries, Osaka, Japan) at an enzyme/substrate ratio of 1:100 for 3 h at 30 ◦C. The resulting Lys-C digested proteins were then digested with TPCKtreated trypsin (Promega, Madison, WI, USA) at an enzyme/substrate ratio of 1:100 for 16 h at 37 ◦C. After a C18 clean-up, the digested protein sample was injected into a NanoLC Ultra system (Eksigent Technologies, Dublin, CA, USA) coupled with an electrosprayionization Triple TOF 5600 mass spectrometer (SCIEX, Framingham, MA, USA), which was set up for single direct injection, and analyzed by SWATH-MS acquisition. The details of measurement and subsequent data analysis have been described previously [17,18]. Finally, the relative expression levels among the control, ssPalm-LNP-treated, and LFN-treated groups were determined for all the quantified proteins. Benjamini–Hochberg adjusted *p* values were calculated, and based on a cutoff of 0.05, the issue of whether there is a significant difference in protein expression level between the groups was determined.

#### **3. Results**

#### *3.1. The mRNA Introduced by the ssPalm-LNP Is Homogeneously Translated in hCMEC/D3 Cells*

The ssPalm-LNPs that were manually prepared by the vortex method had an average particle size of 106.0 ± 15.0 nm, and polydispersity index was 0.09 ± 0.02 in PBS. The ζpotential of −1.5 ± 1.0 mV in 10 mM HEPES buffer (pH 7.4). The distribution in particle size is shown in Figure S2A and indicates that particles with homogenous size distribution were successfully prepared. The mRNA recovery rate was 91.7 ± 12.8%, and the encapsulation efficiency was 86.5 ± 6.1%. This was comparable to a previous report using in vitro a transcribed mRNA encoding luciferase [12]. Considering LFN, size distribution is also homogenous (polydispersity index 0.17 ± 0.03) but slightly large (diameter 340.5 ± 2.5 nm) in PBS (Figure S2B), indicating colloidal formulation of LFN/mRNA complex is also stable even in an isotonic condition.

GFP expression was initially observed by microscopy (Figure 1). Most of the cells treated with the ssPalm-LNPs expressed GFP (Figure 1 left), while only a small fraction of cells was positive in the LFN-treated group (Figure 1 right). To quantitatively evaluate the heterogeneity in gene expression, GFP fluorescence was analyzed by flow cytometry (Figure 2). The mean for the GFP fluorescence intensity in the ssPalm-LNP-treated cells was significantly higher than that in the LFN-treated cells. Of note, the ssPalm-LNP induced GFP expression in a majority (95.0 ± 2.1%) of the cells (Figure 2C), consistent with the microscopic observations. On the other hand, the LFN treatment resulted in the GFP expression in a small fraction of cells (0.43 ± 0.27%). In conclusion, the ssPalm-LNPs showed a drastically higher transfection in hCMEC/D3 cells compared to LFN. GFP expression was initially observed by microscopy (Figure 1). Most of the cells treated with the ssPalm-LNPs expressed GFP (Figure 1 left), while only a small fraction of cells was positive in the LFN-treated group (Figure 1 right). To quantitatively evaluate the heterogeneity in gene expression, GFP fluorescence was analyzed by flow cytometry (Figure 2). The mean for the GFP fluorescence intensity in the ssPalm-LNP-treated cells was significantly higher than that in the LFN-treated cells. Of note, the ssPalm-LNP induced GFP expression in a majority (95.0 ± 2.1%) of the cells (Figure 2C), consistent with the microscopic observations. On the other hand, the LFN treatment resulted in the GFP expression in a small fraction of cells (0.43 ± 0.27%). In conclusion, the ssPalm-LNPs showed a drastically higher transfection in hCMEC/D3 cells compared to LFN. treated with the ssPalm-LNPs expressed GFP (Figure 1 left), while only a small fraction of cells was positive in the LFN-treated group (Figure 1 right). To quantitatively evaluate the heterogeneity in gene expression, GFP fluorescence was analyzed by flow cytometry (Figure 2). The mean for the GFP fluorescence intensity in the ssPalm-LNP-treated cells was significantly higher than that in the LFN-treated cells. Of note, the ssPalm-LNP induced GFP expression in a majority (95.0 ± 2.1%) of the cells (Figure 2C), consistent with the microscopic observations. On the other hand, the LFN treatment resulted in the GFP expression in a small fraction of cells (0.43 ± 0.27%). In conclusion, the ssPalm-LNPs showed a drastically higher transfection in hCMEC/D3 cells compared to LFN.

GFP expression was initially observed by microscopy (Figure 1). Most of the cells

*3.1. The mRNA Introduced by the ssPalm-LNP Is Homogeneously Translated in hCMEC/D3*

*3.1. The mRNA Introduced by the ssPalm-LNP Is Homogeneously Translated in hCMEC/D3*

The ssPalm-LNPs that were manually prepared by the vortex method had an average particle size of 106.0 ± 15.0 nm, and polydispersity index was 0.09 ± 0.02 in PBS. The ζpotential of −1.5 ± 1.0 mV in 10 mM HEPES buffer (pH 7.4). The distribution in particle size is shown in Figure S2A and indicates that particles with homogenous size distribution were successfully prepared. The mRNA recovery rate was 91.7 ± 12.8%, and the encapsulation efficiency was 86.5 ± 6.1%. This was comparable to a previous report using in vitro a transcribed mRNA encoding luciferase [12]. Considering LFN, size distribution is also homogenous (polydispersity index 0.17 ± 0.03) but slightly large (diameter 340.5 ± 2.5 nm) in PBS (Figure S2B), indicating colloidal formulation of LFN/mRNA complex is also stable

The ssPalm-LNPs that were manually prepared by the vortex method had an average particle size of 106.0 ± 15.0 nm, and polydispersity index was 0.09 ± 0.02 in PBS. The ζpotential of −1.5 ± 1.0 mV in 10 mM HEPES buffer (pH 7.4). The distribution in particle size is shown in Figure S2A and indicates that particles with homogenous size distribution were successfully prepared. The mRNA recovery rate was 91.7 ± 12.8%, and the encapsulation efficiency was 86.5 ± 6.1%. This was comparable to a previous report using in vitro a transcribed mRNA encoding luciferase [12]. Considering LFN, size distribution is also homogenous (polydispersity index 0.17 ± 0.03) but slightly large (diameter 340.5 ± 2.5 nm) in PBS (Figure S2B), indicating colloidal formulation of LFN/mRNA complex is also stable

*Pharmaceutics* **2022**, *14*, x 5 of 12

*Pharmaceutics* **2022**, *14*, x 5 of 12

**3. Results**

**3. Results**

even in an isotonic condition.

even in an isotonic condition.

*Cells*

*Cells*

**Figure 1.** GFP expression by mRNA transfection using the ssPalm LNP or LFN. The GFP expression in hCMEC/D3 observed after mRNA transfection at a concentration of 0.4 μg/mL. Nuclei (Hoechst33342) and GFP were depicted in blue and green, respectively. Scale bars: 100 μm. **Figure 1.** GFP expression by mRNA transfection using the ssPalm LNP or LFN. The GFP expression in hCMEC/D3 observed after mRNA transfection at a concentration of 0.4 µg/mL. Nuclei (Hoechst33342) and GFP were depicted in blue and green, respectively. Scale bars: 100 µm. **Figure 1.** GFP expression by mRNA transfection using the ssPalm LNP or LFN. The GFP expression in hCMEC/D3 observed after mRNA transfection at a concentration of 0.4 μg/mL. Nuclei (Hoechst33342) and GFP were depicted in blue and green, respectively. Scale bars: 100 μm.

**Figure 2.** Flow cytometry analysis of GFP expression after mRNA transfection by the ssPalm-LNP or LFN. The GFP expression in hCMEC/D3 cells after mRNA transfection at a concentration of 0.4 µg/mL. (**A**) The representative histogram of the GFP expression in hCMEC/D3 cells. Black, blue, and red lines indicate non-treatment (NT), LFN treatment, and ssPalm-LNP treatment, respectively. (**B**,**C**) The mean GFP fluorescence intensity (**B**) and the percentage of GFP-positive hCMEC/D3 cells (**C**) were analyzed in several independent experiments. Data represent the mean ± standard deviation. Student's t-test was performed between ssPalm and LFN. \*\*\*\*: *p* value < 0.001.

#### *3.2. Higher Amount of the ssPalm-LNP Internalizes hCMEC/D3 Cells Than LFN*

Cellular uptake was also evaluated to determine the reasons for the ssPalm-LNPs being highly expressed in hCMEC/D3 cells. We found that the more homogenous the particles (Figure 3A), the higher the uptake of the ssPalm-LNPs (Figure 3B) in comparison with LFN (Figure 3B). Additionally, the % of DiD-positive cells were also higher in ssPalm-LNP treatment group (Figure 3C).

**Figure 2.** Flow cytometry analysis of GFP expression after mRNA transfection by the ssPalm-LNP or LFN. The GFP expression in hCMEC/D3 cells after mRNA transfection at a concentration of 0.4 μg/mL. (**A**) The representative histogram of the GFP expression in hCMEC/D3 cells. Black, blue, and red lines indicate non-treatment (NT), LFN treatment, and ssPalm-LNP treatment, respectively. (**B,C**) The mean GFP fluorescence intensity (**B**) and the percentage of GFP-positive hCMEC/D3 cells (**C**) were analyzed in several independent experiments. Data represent the mean ± standard devia-

Cellular uptake was also evaluated to determine the reasons for the ssPalm-LNPs being highly expressed in hCMEC/D3 cells. We found that the more homogenous the particles (Figure 3A), the higher the uptake of the ssPalm-LNPs (Figure 3B) in comparison with LFN (Figure 3B). Additionally, the % of DiD-positive cells were also higher in

tion. Student's t-test was performed between ssPalm and LFN. \*\*\*\*: *p* value < 0.001.

*3.2. Higher Amount of the ssPalm-LNP Internalizes hCMEC/D3 Cells Than LFN*

ssPalm-LNP treatment group (Figure 3C).

**Figure 3.** Cellular uptake of the ssPalm LNP or LFN. The cellular uptake of the ssPalm-LNP and LFN by hCMEC/D3 cells was measured by flow cytometry. (**A**) Representative histogram of the uptake of DiD-labeled ssPalm-LNP and LFN by hCMEC/D3 cells at a concentration of 0.4 μg/mL. Black, blue, and red lines indicate the non-treated (NT), the LFN-treated, and the ssPalm-LNPtreated group, respectively. (**B**,**C**) The mean of DiD fluorescence intensity (**B**) and the percentage of DiD-positive hCMEC/D3 cells (**C**) were calculated in several independent experiments. Data represent mean ± standard deviation. Student's *t*-test was performed between ssPalm and LFN. \*\*: *p* value < 0.01. **Figure 3.** Cellular uptake of the ssPalm LNP or LFN. The cellular uptake of the ssPalm-LNP and LFN by hCMEC/D3 cells was measured by flow cytometry. (**A**) Representative histogram of the uptake of DiD-labeled ssPalm-LNP and LFN by hCMEC/D3 cells at a concentration of 0.4 µg/mL. Black, blue, and red lines indicate the non-treated (NT), the LFN-treated, and the ssPalm-LNPtreated group, respectively. (**B**,**C**) The mean of DiD fluorescence intensity (**B**) and the percentage of DiD-positive hCMEC/D3 cells (**C**) were calculated in several independent experiments. Data represent mean ± standard deviation. Student's *t*-test was performed between ssPalm and LFN. \*\*: *p* value < 0.01.

#### *3.3. mRNA Transfection into hCMEC/D3 Cells by ssPalm-LNP Is Significantly Less Cytotoxic Than LFN 3.3. mRNA Transfection into hCMEC/D3 Cells by ssPalm-LNP Is Significantly Less Cytotoxic Than LFN*

To observe the cytotoxicity of the ssPalm-LNP and LFN, hCMEC/D3 cells were observed after treatment at a higher concentration than the above concentrations that were used in these gene expression experiments. In the LFN-treated group, some cells were detached at higher concentrations (Figure 4). The morphology of the cells also differed from that of non-treated cells. At an mRNA concentration of 6.25 μg/mL, these morphological changes were more prominent. In contrast, in the ssPalm-LNP treatment, no change in cell morphology and no detachment of cells were observed, even at the highest concentration (Figure 4). To observe the cytotoxicity of the ssPalm-LNP and LFN, hCMEC/D3 cells were observed after treatment at a higher concentration than the above concentrations that were used in these gene expression experiments. In the LFN-treated group, some cells were detached at higher concentrations (Figure 4). The morphology of the cells also differed from that of non-treated cells. At an mRNA concentration of 6.25 µg/mL, these morphological changes were more prominent. In contrast, in the ssPalm-LNP treatment, no change in cell morphology and no detachment of cells were observed, even at the highest concentration (Figure 4). *Pharmaceutics* **2022**, *14*, x 7 of 12

**Figure 4.** Microscopic observation of hCMEC/D3 cells after the treatment with ssPalm-LNP and LFN. The ssPalm-LNP and the LFN were treated with hCMEC/D3 cells for 48 h at the indicated concentrations. **Figure 4.** Microscopic observation of hCMEC/D3 cells after the treatment with ssPalm-LNP and LFN. The ssPalm-LNP and the LFN were treated with hCMEC/D3 cells for 48 h at the indicated concentrations.

#### *3.4. ssPalm LNP Has Significantly Fewer Adverse Effects Than LFN 3.4. ssPalm LNP Has Significantly Fewer Adverse Effects than LFN*

To evaluate possible effects on off-target proteins, hCMEC/D3 cells were analyzed after being exposed to a higher concentration of the ssPalm-LNP and LFN than used in To evaluate possible effects on off-target proteins, hCMEC/D3 cells were analyzed after being exposed to a higher concentration of the ssPalm-LNP and LFN than used in

the gene transfection experiment. A total of 1899 proteins were quantified by SWATH-MS analysis in the control, ssPalm-LNP-, and LFN-treated groups (Table S1). The levels of

GLUT1, MDR1, and ZO-1) were unchanged among these three groups (Figure S3). Out of 1899 proteins, the significantly up- and down-regulated proteins in the ssPalm-LNPtreated group against the control group were 14 and 26 molecules, respectively (Figure 5). In contrast, the proteins that were significantly up- and down-regulated in the LFNtreated group compared to the control group were 7 and 77 molecules, respectively (Figure 5). This suggests that the LFN treatment results in a more prominent perturbation (mainly decreased) in protein expression, whereas this was minimal in the case of the

**Figure 5.** Volcano plot representation for the protein expression levels of all the quantified proteins by SWATH-MS analysis. The ssPalm-LNP (**A**) and LFN (**B**) were treated with hCMEC/D3 cells for 48 h at a concentration of 2.5 μg/mL. Then, the SWATH-MS analysis was performed for the whole cell lysate of hCMEC/D3 cells. The levels of expression of all the quantified proteins were compared

ssPalm-LNP treatment.

concentrations.

the gene transfection experiment. A total of 1899 proteins were quantified by SWATH-MS analysis in the control, ssPalm-LNP-, and LFN-treated groups (Table S1). The levels of expression of four typical proteins in cerebral vascular endothelial cells (PECAM1, GLUT1, MDR1, and ZO-1) were unchanged among these three groups (Figure S3). Out of 1899 proteins, the significantly up- and down-regulated proteins in the ssPalm-LNP-treated group against the control group were 14 and 26 molecules, respectively (Figure 5). In contrast, the proteins that were significantly up- and down-regulated in the LFN-treated group compared to the control group were 7 and 77 molecules, respectively (Figure 5). This suggests that the LFN treatment results in a more prominent perturbation (mainly decreased) in protein expression, whereas this was minimal in the case of the ssPalm-LNP treatment. the gene transfection experiment. A total of 1899 proteins were quantified by SWATH-MS analysis in the control, ssPalm-LNP-, and LFN-treated groups (Table S1). The levels of expression of four typical proteins in cerebral vascular endothelial cells (PECAM1, GLUT1, MDR1, and ZO-1) were unchanged among these three groups (Figure S3). Out of 1899 proteins, the significantly up- and down-regulated proteins in the ssPalm-LNPtreated group against the control group were 14 and 26 molecules, respectively (Figure 5). In contrast, the proteins that were significantly up- and down-regulated in the LFNtreated group compared to the control group were 7 and 77 molecules, respectively (Figure 5). This suggests that the LFN treatment results in a more prominent perturbation (mainly decreased) in protein expression, whereas this was minimal in the case of the ssPalm-LNP treatment.

**Figure 4.** Microscopic observation of hCMEC/D3 cells after the treatment with ssPalm-LNP and LFN. The ssPalm-LNP and the LFN were treated with hCMEC/D3 cells for 48 h at the indicated

To evaluate possible effects on off-target proteins, hCMEC/D3 cells were analyzed after being exposed to a higher concentration of the ssPalm-LNP and LFN than used in

*Pharmaceutics* **2022**, *14*, x 7 of 12

*3.4. ssPalm LNP Has Significantly Fewer Adverse Effects Than LFN*

**Figure 5.** Volcano plot representation for the protein expression levels of all the quantified proteins by SWATH-MS analysis. The ssPalm-LNP (**A**) and LFN (**B**) were treated with hCMEC/D3 cells for 48 h at a concentration of 2.5 μg/mL. Then, the SWATH-MS analysis was performed for the whole cell lysate of hCMEC/D3 cells. The levels of expression of all the quantified proteins were compared **Figure 5.** Volcano plot representation for the protein expression levels of all the quantified proteins by SWATH-MS analysis. The ssPalm-LNP (**A**) and LFN (**B**) were treated with hCMEC/D3 cells for 48 h at a concentration of 2.5 µg/mL. Then, the SWATH-MS analysis was performed for the whole cell lysate of hCMEC/D3 cells. The levels of expression of all the quantified proteins were compared with those in the control group (*n* = 5–9). X-axis represents the log2 values for the fold changes in protein expression levels compared with the control group. Y-axis represents the minus log10 values of the Benjamini–Hochberg (BH) adjusted *p* values for the differences between two groups. Blue and red areas represent the down- and up-regulated proteins with *p* values less than 0.05.

In the above experiments, unlike LFN, the ssPalm-LNPs did not cause cytotoxicity. To understand the molecular mechanisms that are involved in this difference, proteins with significant differences in expression between the LFN and control groups, but not between the ssPalm and control groups, were extracted. As a result, 67 proteins were extracted (Table S2). These were analyzed by a "String functional protein association network" analysis, and as the top two clusters, translation-related proteins and chaperonincontaining TCP-1 (CCT) (Figure 6) were then identified. As translation-related proteins, the levels of expression of RPS3, RPL11, RPL27, EIF2S3, EIF3F, EFTUD2, and HSPD1 proteins were significantly decreased by the LFN treatment by 1.17-, 1.17-, 1.28-, 1.20-, 1.27-, 1.10-, and 1.12-fold, respectively, whereas no significant reduction was observed in ssPalm-LNPtreated group (Figure 6). As CCTs, the levels of expression of the TCP1, CCT2, CCT3, and CCT4 proteins were significantly reduced by the LFN treatment by 1.15-, 1.13-, 1.10-, and 1.16-fold, respectively, but not significantly in the ssPalm-LNP-treated group (Figure 6).

**Figure 6.** Top 2 clusters that are down-regulated in the LFN group but not in the ssPalm group. (**A**) 67 proteins whose expression levels were significantly changed in the LFN-treated group but not in the ssPalm-LNP-treated group (Table S2 analyzed using the STRING Database (https://string-db.org/ accessed on 2 May 2022) to visualize functional protein association networks [19] with an MCL clustering mode based exclusively on experimentally determined interactions. For the top 2 clusters, the interaction maps obtained on the String website are shown in this figure. The first cluster consists of translation-related proteins (red-colored nodes), and the second cluster consists of chaperonincontaining TCP-1 (CCT) proteins (green-colored nodes). (**B**,**C**) the levels of expression of CCT (**B**) and translation-related proteins (**C**) were compared among the control, ssPalm-LNP-treated group, and LFN-treated groups (*n* = 5–9). The band inside the box represents the median, and the bottom and top of the box indicate the first and third quartiles, respectively. Whiskers indicate the minimum and maximum values of the protein levels. X plots show the average in each group. \* the Benjamini– Hochberg adjusted *p* value < 0.05 was significantly down-regulated compared to control group. N.S.—not significantly different (Benjamini–Hochberg adjusted *p* value > 0.05).

#### **4. Discussion**

1

In the present study, we reported that mRNA-loaded ssPalm-LNPs can be used to efficiently transfect hCMEC/D3 cells with a marker gene GFP without any obvious toxicity. Although previous reports have not provided a clear reason for the low transfection

efficiency in BBB-derived endothelial cells, the present results suggest that one major factor is the low uptake of cationic substrates (Figure 3). Commonly used cancer cells and mouse embryo fibroblasts exhibited a much higher uptake of cationic nanoparticles compared to neutral ones [7,20,21]. This high uptake of cationic substances is thought to be, at least in part, due to interactions with negatively charged proteoglycans on the outer surface of the cell membrane [22]. Although the exact mechanism is unclear, it is possible that this uptake pathway might not be available in hCMEC/D3 cells. On the other hand, the uptake of neutral LNPs, including ssPalm-LNPs, is attributed to the formation of complexes of LNPs with Apoproteins (Apos) in the culture medium or in the biological fluid, and apolipoprotein receptors such as low-density lipoprotein receptors (LDLR) subsequently recognize these complexes [23–25]; it has been reported that LDLR and lowdensity lipoprotein receptor-related protein (LRP), a receptor for Apos, are expressed in BMEC [26,27]. The current SWATH-MS results showed the expression of LRP2, LRP8, and various apoproteins such as apoproteins A-I, B, E, and L3 (Table S1), although the amounts are not currently known. Taken together, the uptake of ssPalm-LNP may have been much higher than that of LFN due to the interaction of these receptors in hCMEC/D3 with medium-derived Apos that are adsorbed on the LNPs. In addition, it should be noted that the mRNA localization and whole structure of these nanoparticles were completely different: mRNA would be encapsulated into lipid molecules (LNP), or mRNA would be attached to the cationic surface of liposomes (LFN). Actually, the size of the ssPalm LNP was smaller than LFN (106.0 nm vs. 340.5 nm). The effect of the difference in the size and structure on the gene transfection efficacy should be taken into account.

The ssPalm-LNPs did not alter the morphology of the hCMEC/D3 cells (Figure 4), even though cells were treated with the ssPalm-LNP at 15.6-fold higher concentrations than that for the controls where >95% of the cells were transfected (Figures 1 and 2). Furthermore, compared to the LFN, the ssPalm-LNPs had less adverse effects on cellular protein expression (Figure 5). These data suggest that the ssPalm-LNPs developed in this study are superior for mRNA delivery to brain capillary endothelial cells in terms of cytotoxicity and off-target effects. It should also be noted that the levels of expression of many proteins were significantly down-regulated as the result of the LFN treatment (Figure 5). This is consistent with a report showing that the expression of various proteins was reduced in Lipofectamine 2000-treated HUVEC cells [6]. This may be attributed to the significant reduction in protein translation, such as ribosomal proteins and chaperonin (HSPD1/HSP60 and CCT/TRiC) in the LFN-treatment group (Figure 6).

When misfolded proteins accumulate in the cell, the unfolded protein response (UPR) is activated at the endoplasmic reticulum (ER) to reduce the accumulation of misfolded proteins. If the UPR fails to restore the ER to normality, ER stress can promote apoptosis [6]. In HUVEC cells, lipofectamine 2000 causes UPR and suppresses cell proliferation [6]. CCT/TRiC and HSPD1/HSP60 are two important chaperonins that interact with misfolded proteins to prevent misfolding and aggregation and facilitate correct folding. These levels of expression of these proteins were reduced in the case of the LFN treatment in hCMEC/D3 cells (Figure 6). This may have resulted in an over-accumulation of misfolded proteins in the cells, causing cell death. Since ssPalm-LNPs do not adversely affect the expression of molecules that are involved in protein translation and chaperonins (HSPD1/HSP60 and CCT/TRiC), they would be expected to be safe materials for future in vivo gene delivery to brain capillary endothelial cells.

The currently reported mRNA transfection technology could be applied to the expression of various genes. We demonstrated the successful encapsulation of mRNA molecules ranging from 850 bases to more than 4.5 kilobases regardless of the presence or absence of chemical modification (i.e., N1-Methylpseudouridine) [7]. Additionally, the introducible proteins are not limited to cytosolic proteins such as GFP: membrane proteins such as transporters and tight junction-related proteins can also be expressed. Further, although we demonstrate herein the efficient gene transfection with monocultured, non-polarized brain capillary endothelial cells, the mRNA transfection by the ssPalm-LNP is expected

to be applied for multiplexed culture (endothelial cells, astrocytes, pericytes, and related cells), which are currently used as in vivo BBB model [28,29] since the LNP can transfect mRNA even in complete medium containing serum.

#### **5. Conclusions**

We report herein that the ssPalm-LNP is a promising carrier for efficiently transporting mRNA into the non-polarized human brain capillary endothelial cells, hCMEC/D3. The transfection efficiency of the ssPalm LNP was found to be much higher than that of a commercially available transfection reagent Lipofectamine MessengerMAX. Treatment with high concentrations of the ssPalm-LNP did not induce cytotoxicity in comparison with LFN. Further, a SWATH-MS analysis revealed that the exposure of hCMEC/D3 cells to the ssPalm-LNP had only minimal effects on cellular proteins. These results indicate that the ssPalm-LNP represents a potent tool for elucidating the functional, biological chrematistics of the brain endothelium by the transfection of a gene of interest.

**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/pharmaceutics14081560/s1, Figure S1: The structures of lipid molecules used in the ssPalm LNP; Figure S2: The particle size distribution of ssPalm LNP; Figure S3: Protein expression levels of BBB marker proteins in hCMEC/D3 cells treated with ssPalm LNP and Lipofectamine; Table S1: Relative protein expression levels of all the proteins quantified by SWATH-MS analysis among control, ssPalm LNP, and lipofectamine groups; Table S2: The proteins whose expression levels were significantly changed in Lipofectamine group but not changed in ssPalm group.

**Author Contributions:** Conceptualization, Y.S., H.A. and Y.U.; methodology, Y.S., M.G., M.S., R.O. and Y.U.; software, Y.S., M.G., M.S., R.O. and Y.U.; validation, H.W., K.N., K.H., A.H., M.G. and M.S.; formal analysis, H.W., K.N., K.H. and A.H.; investigation, H.W., K.N., K.H., A.H., T.H., R.S., H.T., R.T. and K.T. (Kenta Tezuka); resources, K.T. (Kota Tange) and Y.N.; data curation, H.W., K.N., K.H., A.H., M.S. and Y.U.; writing—original draft preparation, Y.S. and Y.U.; writing—review and editing, Y.S., H.A. and Y.U.; visualization, Y.S. and Y.U.; supervision, Y.S., H.A. and Y.U.; project administration, Y.S. and Y.U.; funding acquisition, Y.S., H.A. and Y.U. All authors have read and agreed to the published version of the manuscript.

**Funding:** The study was supported, in part, by the JST core research for evolutional science and technology (crest) grant (grant number: JPMJCR17H1), the JSPS KAKENHI Grant-in-Aid for Scientific Research (B) (grant number: 20H03399), a Grant-in-Aid for Young Scientists (grant number: 20K20195), the MEXT KAKENHI Grant-in-Aid for Scientific Research on Innovative Areas (grant number: 22H04798).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available from the corresponding authors on reasonable request.

**Acknowledgments:** The authors wish to thank Pierre-Olivier Couraud for generously providing the hCMEC/D3 cell line (Institut Cochin, France) and Milton S Feather (a scientific editor in the United States of America) for English proofreading of the entire manuscript.

**Conflicts of Interest:** This research was conducted as joint research between Tohoku University and NOF CORPORATION. The other funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

#### **References**

## *Review* **Modeling Blood–Brain Barrier Permeability to Solutes and Drugs In Vivo**

**Ulrich Bickel 1,2**

**Abstract:** Our understanding of the pharmacokinetic principles governing the uptake of endogenous substances, xenobiotics, and biologicals across the blood–brain barrier (BBB) has advanced significantly over the past few decades. There is now a spectrum of experimental techniques available in experimental animals and humans which, together with pharmacokinetic models of low to high complexity, can be applied to describe the transport processes at the BBB of low molecular weight agents and macromolecules. This review provides an overview of the models in current use, from initial rate uptake studies over compartmental models to physiologically based models and points out the advantages and shortcomings associated with the different methods. A comprehensive pharmacokinetic profile of a compound with respect to brain exposure requires the knowledge of BBB uptake clearance, intra-brain distribution, and extent of equilibration across the BBB. The application of proper pharmacokinetic analysis and suitable models is a requirement not only in the drug development process, but in all of the studies where the brain uptake of drugs or markers is used to make statements about the function or integrity of the BBB.

**Keywords:** pharmacokinetics; blood–brain barrier; compartmental models; physiologically based PK models

#### **1. Introduction**

It has been over 120 years since the earliest publications on the distinct features of the brain vasculature compared to other organs, which over time gave rise to the concept of a "blood–brain barrier" (BBB). As recently reviewed [1], there have been many misconceptions along the way about its structure and function. It is now established that the physical and biochemical BBB is formed by the endothelial cells of the brain microvasculature, which are connected by tight junctions (Figure 1). The barrier function is induced or modulated by other elements of the "neurovascular unit" [2], including pericytes, astrocytes, neurons, and microglial cells. Some misunderstandings linger to this day in parts of the literature. One area concerns the methods of measuring the brain uptake of drugs and the proper evaluation and interpretation. The present review will provide an overview of the development and application of modeling approaches to describe the pharmacokinetics (PK) of brain uptake of low molecular weight drug-like solutes and macromolecules. We will not attempt to include all of the aspects of the extensive and rapidly growing literature in this field, but aim to focus on practical applicability with some examples.

The purpose of the PK models covers a range from the evaluation and the fitting of sets of experimental data that are as good as possible to simulations and predictions based on few or no experimental data, to employing complex physiologically based pharmacokinetic (PBPK) models with parameter values obtained from in silico, in vitro, and in vivo studies. Which model is appropriate also depends on the experimental design in each case and

**Citation:** Bickel, U. Modeling Blood–Brain Barrier Permeability to Solutes and Drugs In Vivo. *Pharmaceutics* **2022**, *14*, 1696. https://doi.org/10.3390/ pharmaceutics14081696

Academic Editor: Paul Chi Lui Ho

Received: 8 July 2022 Accepted: 10 August 2022 Published: 15 August 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the author. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

the physicochemical characteristics of the agent to be evaluated. Macromolecules differ greatly in kinetic behavior from small molecule drugs and studying poorly permeable substances requires a different experimental design and PK evaluation approaches than highly permeable drugs. From an experimental perspective, it is feasible in preclinical studies to obtain blood and (brain) tissue samples at multiple time points, while clinical studies are typically limited to blood sampling, and occasionally CSF sampling, and noninvasive imaging modalities (MRI, SPECT, PET), if applicable. Finally, a distinction can be made, based on whether the parameter of primary interest is the uptake rate across the BBB or the brain exposure (fraction of a dose, or extent). These models will be presented, from the simple to the more complex. *Pharmaceutics* **2022**, *14*, x 2 of 18

**Figure 1.** Scheme of the BBB: The endothelial cells of the capillaries are connected by tight junctions and form a physical and biochemical barrier. The pericytes and astrocytes play critical roles in the induction and maintenance of the endothelial barrier properties. Microglial cells and neurons also secrete signals, which can influence the endothelial cells. The diameter of the capillaries is on the order of 7–10 µm. Figure adapted from Reference [3] with permission. Copyright 2010, Elsevier. **Figure 1.** Scheme of the BBB: The endothelial cells of the capillaries are connected by tight junctions and form a physical and biochemical barrier. The pericytes and astrocytes play critical roles in the induction and maintenance of the endothelial barrier properties. Microglial cells and neurons also secrete signals, which can influence the endothelial cells. The diameter of the capillaries is on the order of 7–10 µm. Figure adapted from Reference [3] with permission. Copyright 2010, Elsevier.

#### The purpose of the PK models covers a range from the evaluation and the fitting of sets of experi-**2. Models Assuming Unidirectional Brain Uptake**

mental data that are as good as possible to simulations and predictions based on few or no experimental data, to employing complex physiologically based pharmacokinetic (PBPK) models with parameter values obtained from in silico, in vitro, and in vivo studies. Which model is appropriate also depends on the experimental design in each case and the physicochemical characteristics of the agent to be evaluated. Macromolecules differ greatly in kinetic behavior from small molecule drugs and studying poorly permeable substances requires a different experimental design and PK evaluation approaches than highly permeable drugs. From an experimental perspective, it is feasible in As the name implies, unidirectional organ uptake models are based on the concept that for the duration of the experiment, a drug or tracer diffuses (or is undergoing transport) only from the blood side (luminal) to the brain side (abluminal) across the BBB. Provided that there is no significant backflux, it allows the determination of the initial rate of brain uptake. The approach can be most readily illustrated considering the examples of carotid injection and carotid artery brain perfusion techniques.

preclinical studies to obtain blood and (brain) tissue samples at multiple time points, while clinical

#### studies are typically limited to blood sampling, and occasionally CSF sampling, and noninvasive *2.1. Brain Uptake Index (BUI)*

imaging modalities (MRI, SPECT, PET), if applicable. Finally, a distinction can be made, based on whether the parameter of primary interest is the uptake rate across the BBB or the brain exposure (fraction of a dose, or extent). These models will be presented, from the simple to the more complex. **2. Models Assuming Unidirectional Brain Uptake**  As the name implies, unidirectional organ uptake models are based on the concept The technique, as introduced by Oldendorf [4,5], uses radiotracers labeled with different isotopes and measures the brain tissue-extraction ratio (brain concentration: injectate concentration) of a compound during a single pass through the cerebral vasculature after bolus injection into the carotid artery under anesthesia. BUI (as a percentage) is then estimated from the brain extraction ratio of the test substance (*Etest*), relative to that of a permeable reference substance (*ErefP*) with a known, near total extraction:

that for the duration of the experiment, a drug or tracer diffuses (or is undergoing

of brain uptake. The approach can be most readily illustrated considering the examples of

$$BIII = \frac{E\_{\text{test}}}{E\_{\text{refP}}} \times 100 = \frac{\text{C}\_{\text{test}} \text{in } b \text{rain/C}\_{\text{test}} \text{in} \text{ in} \text{jetstate}}{\text{C}\_{\text{refP}} \text{in } b \text{rain/C}\_{\text{refP}} \text{in} \text{ in} \text{jetstate}} \times 100\tag{1}$$

௧௦௧ ௧௦௧ ⁄

/ × 100 (1)

carotid injection and carotid artery brain perfusion techniques. *2.1. Brain Uptake Index (BUI)*  The technique, as introduced by Oldendorf [4,5], uses radiotracers labeled with different isotopes and measures the brain tissue-extraction ratio (brain concentration: injecwhere *Ctest* refers to the concentrations of the test substance and *CrefP* refers to the concentrations of the permeable reference. The examples of highly permeable substances with a complete (100%) extraction include iodoamphetamine [6] and diazepam [7]. In order to correct for brain intravascular volume, an additional reference substance was introduced, which is assumed to show no tissue uptake during the single passage.

tate concentration) of a compound during a single pass through the cerebral vasculature after bolus injection into the carotid artery under anesthesia. BUI (as a percentage) is then estimated from the brain extraction ratio of the test substance (*Etest*), relative to that of a

where *Ctest*refers to the concentrations of the test substance and *CrefP* refers to the concentrations of the permeable reference. The examples of highly permeable substances with a complete (100%) extraction include iodoamphetamine [6] and diazepam [7]. In order to correct for brain intravascular volume, an additional reference substance was introduced,

௧௦௧

=

permeable reference substance (*ErefP*) with a known, near total extraction:

which is assumed to show no tissue uptake during the single passage.

*BUI* in percent is then calculated as the ratio:

$$BIII = 100 \times \left( E\_{test} - E\_{ref\,V} \right) / E\_{ref\,P} \tag{2}$$

where *ErefV* is the apparent "extraction" of the vascular marker (non-permeant reference). *Etest* can then be used to calculate the permeability surface area product (*PS*), applying a rearranged Renkin–Crone equation:

$$PS = -F \ln(1 - E) \tag{3}$$

where *F* is the tissue perfusion flow rate. The BUI method allows wide flexibility in the composition of the injectate and the concentrations of the test substance. In the BUI technique, the brain tissue is treated as one compartment. It has been successfully applied to study, in particular, the BBB transport of nutrients, including the characterization of saturable transport by applying Michaelis–Menten kinetics [8]. A limitation of the technique is its low sensitivity for poorly permeable solutes. For example, the brain uptake of the classical CNS-active drug, morphine could not be distinguished from [14C]sucrose, which behaves as a vascular marker in a single-pass experiment [9].

#### *2.2. In Situ Brain Perfusion*

These techniques perfuse the brain in anesthetized animals with oxygenated buffer solutions via the carotid artery. Similar methods have been established in the rat [7], guinea pig [10], and mouse [11,12] and are in widespread use. The pharmacokinetic principle is analogous to the BUI method, in the assumption that the initial rate of brain uptake can be measured as long as the effective tissue concentrations are much lower than the perfusate concentration and unidirectional transport prevails. The evaluation is based on a plot of the apparent volume of distribution (*VD*) in the brain compartment against perfusion time (*T*), where *VD* is the ratio of the amount of the test substance in the brain per unit weight, *Abr* (e.g., nmol/g brain) over the perfusate concentration (*Cperf*) of the analyte (e.g., nmol/mL), with *Cperf* being constant. With a unidirectional uptake, this results in a linear increase, and the slope of a linear regression line represents the unidirectional transfer constant, *Kin*, from the perfusate fluid into the brain compartment, which is a clearance parameter, e.g., in units of [mL/min]:

$$V\_D = \frac{A\_{br}}{\mathcal{C}\_{perf}} = K\_{in}T + V\_{\bar{l}} \tag{4}$$

The y-intercept of the regression line represents an "initial" volume of distribution, *Vi* . It corresponds to the sum of the intravascular volume, *V*0, which in rodents typically is in a range of 8–10 µL/g [13], and a potential residual volume, which is physiologically difficult to characterize, e.g., caused by nonspecific binding to the vascular wall, or associated with cellular components of the BBB. In cases where substantially higher values of *Vi* are observed, the inclusion of a marker substance, such as albumin or inulin, which is expected to undergo negligible transport across the BBB, can be applied to experimentally measure the intravascular space. *Kin* approaches the value of the PS product at the BBB within 10% for conditions of permeability-limited transport (low E), when flow F is greater than 5x PS. Depending on the test compound, in situ perfusions are typically conducted over a few seconds up to several minutes. Similar to BUI, the experimental advantages are that the perfusate compositions and concentrations of the test substance can be controlled within wide limits, outside of the conditions tolerated in the whole animal with systemic administration. On the other hand, the sensitivity for accurate permeability measurements of analytes with low permeability is superior to the single-pass technique, but is still limited. The extended perfusion times (e.g., 10–20 min) require the inclusion of oxygen carriers, such as washed erythrocytes in the perfusate, to avoid hypoxic conditions [10].

#### *2.3. Intravenous Injection*

The most physiological technique for the analysis of BBB transport is the intravenous administration (as a bolus or infusion), followed by the analysis of the concentration time course in blood and brain. To obtain reliable and correct measurements, several conditions have to be met in such studies. First, considering the free drug hypothesis [14], the free fraction in plasma needs to be determined by microdialysis in vivo, or by ex vivo techniques, such as ultrafiltration. This will be discussed in more detail below in Section 4. Second, if metabolism occurs, the analytical method used must be able to measure the intact substance in the plasma and tissue.

#### 2.3.1. Multiple Time Point Analysis

Depending on the experimental design, different options for the pharmacokinetic evaluation of brain uptake are available. With a series of brain and plasma samples taken at different time points, the multiple time-point graphical evaluation, also known as a Patlak plot, is frequently used [15–17]. In its original form, it modeled the brain tissue as consisting of several reversible compartments and one irreversible compartment [17]. A practical example is the intracellular entrapment of tracers, such as deoxy-glucose or α-amino-isobutyric acid. Under the condition that the reversible compartments are in rapid exchange with the plasma compartment, Patlak and colleagues showed that the unidirectional brain uptake can be analyzed by plotting the time-dependent apparent volume of distribution against the ratio of the plasma area under the curve (AUC) from time zero to time T and terminal plasma concentration at time T:

$$\frac{A\_{br}(T)}{\mathbb{C}\_p(T)} = K\_{in} \frac{\int\_0^T \mathbb{C}\_p(t)dt}{\mathbb{C}\_p(T)} + V\_{\bar{l}} \tag{5}$$