Exploring and Analyzing the Systemic Delivery Barriers for Nanoparticles

Abstract

Most nanomedicines require efficient in vivo delivery to elicit diagnostic and therapeutic effects. However, en route to their intended tissues, systemically administered nanoparticles often encounter delivery barriers. To describe these barriers, we propose the term "nanoparticle blood removal pathways" (NBRP), which summarizes the interactions between nanoparticles and the body's various cell-dependent and cell-independent blood clearance mechanisms. We reviewed nanoparticle design and biological modulation strategies to mitigate nanoparticle-NBRP interactions. As these interactions affect nanoparticle delivery, we studied the preclinical literature from 2011-2021 and analyzed nanoparticle blood circulation and organ biodistribution data. Our findings revealed that nanoparticle surface chemistry affected the in vivo behavior more than other nanoparticle design parameters. Combinatory biological-PEG surface modification improved the blood area under the curve by ~418%, with a decrease in liver accumulation of up to 47%. A greater understanding of nanoparticle-NBRP interactions and associated delivery trends will provide new nanoparticle design and biological modulation strategies for safer, more effective, and more efficient nanomedicines.

Keywords: Nanomedicine; biodistribution; biological barriers; literature survey; mononuclear phagocyte system (MPS); nano-bio interaction; nanoparticle blood removal pathways (NBRP); nanoparticle delivery; pharmacokinetics; reticuloendothelial system (RES).

Figure 1:. Examples of nanoparticle delivery barriers.

a. The body’s biological and physical barriers affect nanoparticle biodistribution. Note: Depicted relative biodistribution and nanoparticle accumulation may vary significantly for different nanoparticle formulations and doses. b. Upon intravenous administration (1), nanoparticles are exposed to blood components. This exposure changes the nanoparticle’s synthetic identity to a biological identity. During the initial exposure phase (2), proteins with varying binding affinities interact dynamically with the nanoparticle surface. Over time (3), a hard protein corona forms around the nanoparticle surface composed of proteins exhibiting relatively high binding affinity and low exchange rate. Proteins with less affinity and high exchange rate interact with the hard corona and create a dynamic soft corona. c. Cells of the nanoparticle blood removal pathways (NBRP), including tissue-resident macrophages, circulating leukocytes, and various endothelial cell types, uptake circulating nanoparticles from the blood by various mechanisms.

Figure 2:. Schematic overview of the Nanoparticle Blood Removal Pathways (NBRP).

The NBRP is a broadly defined term that summarizes multiple mechanisms of nanoparticle clearance and sequestration. The two main branches of the NBRP are the cell-dependent and the cell-independent NBRP. The cell-dependent NBRP include traditional systems used to describe nanoparticle clearance, such as the MPS and RES, and further incorporates other cell-mediated pathways, such as clearance by other leukocytes and additional unknown/undiscovered pathways and mechanisms. The cell-independent NBRP include physical clearance mechanisms, such as glomerular filtration in the kidneys and the sinusoids of the liver and spleen. As additional mechanisms of nanoparticle clearance may be discovered in the future, these mechanisms will be adequately described by the proposed NBRP terminology.

Figure 3:. Schematic of the liver micro-architecture.

a. The liver is composed almost homogeneously of microscopic functional units called liver lobules (b). Blood flows into the liver via the hepatic veins and arteries. Together with the bile duct, these two vessels form the portal triad. Blood flows from each portal triad to the three nearest central veins, and bile flows in the opposite direction to collect in the bile duct for excretion. The classic lobule model comprises a hexagon tracing the six nearest portal triads surrounding a given central vein. The portal lobule model is visualized with a triangle connecting three adjacent central veins. The acinus model is described as a diamond shape with two portal triads on the short axis and two central veins on the long axis. The acinus model is most relevant to nanoparticle clearance as it emphasizes blood flow from the portal triads and the vessel network connecting them to the central vein. As blood flows toward the central vein, it passes through small vessels called sinusoids (c). Blood velocity decreases significantly, and the blood is allowed to interact with various cell types. As shown in panel (c), nanoparticles in the blood interact with different cell types, including Kupffer cells (macrophages), fenestrated liver sinusoidal endothelial cells (LSECs), and hepatocytes. The liver sinusoids are the primary location in the body where clearance of foreign materials (including engineered nanoparticles) from the blood occurs.

Figure 4:. Schematic of the spleen micro-architecture.

a. The spleen is fed by arterial blood through the splenic artery and drains through the splenic vein. Arterial blood flows through arterioles and out into the white and red pulp of the spleen. b. A small amount of blood is processed in the white pulp, where the spleen’s complex lymphoid (adaptive immunity) function is carried out. Most blood flows out of the branched arterioles into the red pulp of the spleen. The blood is pushed into the splenic cords and collects in the splenic sinuses to exit the spleen. c. The splenic sinuses comprise unique, lengthened endothelial cells with parallel stress fibers and perpendicular annular fibers contributing to the spleen’s filtering function. (1) Venous blood containing nanoparticles enters the splenic cords through arterioles. (2) Blood and nanoparticles flow through the splenic cords until pressure pushes them against the slits between the endothelial cells of the sinus. (3a) Nanoparticles or old RBCs that are too large or inflexible to pass through the slits will persist in the splenic cords and eventually be cleared by red pulp macrophages, B cells, and dendritic cells. (3b) Nanoparticles and healthy RBCs that are small or flexible enough to pass through the slits will exit the splenic sinus and return to circulation.

Figure 5:. Schematic of the lung micro-architecture.

a. The lung has the largest vasculature system in the human body, bringing about a vast endothelium surface area of almost 70 m² for the interaction between nanoparticles and endothelial cells. b. The lung is composed of more than 300 million alveoli. Each alveolus is covered with numerous pulmonary capillaries, forming the functional unit for gas exchange. c. Nanoparticles interact with different lung cells after systemic administration. Neutrophils, rather than intravascular macrophages, are the major immune cell type in the lung capillaries. Neutrophils do not significantly internalize nanoparticles without activation. When passing through the lung capillaries, nanoparticles interact with the endothelium. The endothelial cells are tightly aligned along the vascular wall, exhibiting no gaps or fenestrae. In general, only a small portion of systemically administered nanoparticles cross the endothelium and interact with pulmonary macrophages and alveolar epithelial cells.

Figure 6:. Schematic of the bone marrow micro-architecture.

a. The bone is made up of compact bone, spongy bone, and bone marrow. Red bone marrow, where the venous sinuses locate, is mainly found in the spongy bone, while yellow bone marrow is in the central cavity of long bones. b. The primary blood supply to the bone marrow enters through the cortical bone and reaches the central artery in the medulla cavity via the nutrient artery. After meeting the periosteal arterial supply, blood drains into venous sinuses. Venous sinuses are capillary networks that support hematopoietic cells located in the hemopoietic spaces. The sinusoidal endothelium is fenestrated and is covered by an interrupted basement lamina. Mature blood cells can cross the sinusoidal endothelium and enter the bloodstream. Eventually, these sinuses converge to the central sinus, and blood exits the medullary cavity through emissary veins.

Figure 7:. Schematic of kidney micro-architecture.

a. The kidneys receive blood from circulation. Filtered blood exits back into circulation, and the filtrate exits through the ureter to excretion in the urine. b. The glomerulus is the functional unit of the kidney. Blood enters through the afferent arteriole, passes through the glomerular capillaries, and exits through the efferent arteriole. Particles and other waste products that exit the glomerular capillaries are collected in Bowman’s capsule and then exit through the proximal tubule. c. The boundary of the glomerular capillaries comprises the vessel endothelial layer, the glomerular basement membrane, and a layer of podocytes surrounding the vessel. This boundary acts as a filtration membrane that lets nanoparticles through with different dynamics relative to their size and charge characteristics. Generally, positive particles and particles with a diameter of fewer than six nanometers experience rapid clearance and subsequent excretion in the urine.

Figure 8:. Nanoparticle design modulation strategies to reduce NBRP interactions and clearance.

The intrinsic nanoparticle physicochemical properties, including (a) size, (b) shape, (c) elasticity/stiffness, and (d) surface charge, can be modulated to reduce uptake by NBRP organs and cells. e. Nanoparticle surface modifications exhibit a broad range of methods ranging from purely synthetic to biologically-inspired surface modifications.

Figure 9:. Biological modulation strategies.

a. Saturation preconditioning strategies work by overloading NBRP cells with a non-therapeutic nanoparticle, i.e. filler nanoparticle. The nanoparticle used for saturation is usually chosen to be nontoxic and should degrade quickly after a period of time. Since NBRP cells are full of the saturating nanoparticle and/or their uptake rates are saturated, these cells cannot further engulf more nanoparticles, allowing subsequently and/or simultaneously administered therapeutic/diagnostic nanoparticles to evade the NBRP and distribute more effectively to target tissues. b. Endocytosis inhibition strategies use drugs that block the interactions between NBRP cells and nanoparticles. One mechanism is the disruption of endocytosis mechanisms by blocking receptor-nanoparticle corona interactions. Nanoparticles escape attachment to NBRP cell membranes and are free to interact more efficiently with target tissues. c. In the cell suicide strategy, drugs induce cell death in all or part of resident tissue macrophage populations, resulting in reduced nanoparticle sequestration by the NBRP.

Figure 10:. Procedures and filters used for the literature analysis.

Peer-reviewed publications from 2011–2021 were screened and systematically organized based on their reported nanoparticle pharmacokinetics and organ distribution data. A total of 113 adequate papers were identified.

Figure 11.. Analysis of the nanoparticle pharmacokinetics in mice.

a. The non-compartmental model used was the slope, height, area, and momentum (SHAM) model. This model was applied to calculate the area-under-the-curve from the zero point to infinity (AUC_0-infinity) of the plot of nanoparticle concentration in blood versus time. Equations (1–5) were used to calculate AUC_0-infinity, which is composed of two parts, AUC_0-t (AUC from zero to last time point; equations 1–2) and AUC_t-infinity (AUC from last time point to infinity; equations 3–4). AUC_0-t was calculated by the linear trapezoidal method, in which T_i represents the area of the trapezoid between time t_{i – 1} and t_i (equation 1), and AUC_0-t is obtained by summing up all trapezoid areas from i=1 to i=n (equation 2). AUC_t-infinity, the “triangle” at the end of the curve, was calculated using the terminal slope (k_el, elimination rate constant) and the nanoparticle concentration at the last time point (C_last). k_el was obtained based on the data of the last two time points, (C_last, t_last) and (C_last-1, t_last-1), according to equation 3. AUC_t-infinity was then calculated by dividing C_last by k_el (equation 4). Finally, AUC_0-infinity equals the summation of AUC_0-t and AUC_t-infinity (equation 5)_. b-j. Categorical analysis of the blood AUC_0-infinity data. The effects of factors were analyzed, including: Material (b), Inorganic Material composition (c), Organic Material composition (d), Hydrodynamic Diameter (e), Shape (f), Zeta Potential (g), PEG-containing condition (h), Surface Chemistry (i), and Animal Model (j). Individual data points and box-and-whisker plots are presented simultaneously. In the plots, a box represents the 25th to 75th percentile values, and the black solid line in the box indicates the median. The top and bottom lines indicate the maximum and minimum values.

Figure 12.. Categorical analysis of nanoparticle concentration data in the liver 24 hours after systemic administration.

The effects of factors were analyzed, including: Material (a), Inorganic Material composition (b), Organic Material composition (c), Hydrodynamic Diameter (d), Shape (e), Zeta Potential (f), PEG-containing condition (g), Surface Chemistry (h), and Animal Model (i). Individual data points and box-and-whisker plots are presented simultaneously. In the plots, a box represents the 25th to 75th percentile values, and the black solid line in the box indicates the median. The top and bottom lines indicate the maximum and minimum values.

Figure 13.. Proposed workflow to enhance nanomedicine translation.

We propose a workflow to transform the understanding of nano-bio interactions and to advance progress in nanomedicine development and clinical application. PK, pharmacokinetics; BD, biodistribution