1.

Introduction

Diabetes and other debilitating conditions are diagnosed, treated, and monitored by blood sampling. For diabetics, diet, insulin regulation, and glucose monitoring ameliorate serious complications. The primary devices for glucose monitoring, finger stick-based glucose meters, suffer from issues, such as human error and meter/strip quality as well as being susceptible to interfering factors.^1–3 Poor control of blood glucose levels can result in complications affecting the eyes, heart, kidneys, nerves, and feet.⁴ In addition, development of hypoglycemia or hyperglycemia can lead to death. Finger pricking multiple times throughout the day hinders the accurate identification of trends, reduces compliance, and increases the risk of complications.^3,4 Thus, there is an urgent need to explore continuous blood analyte sensing.

Continuous glucose monitoring systems (CGMS) have limited sensing lifetime and must address immunogenicity, selectivity, and biodegradation issues. Current CGMS have lifetimes on the order of 10 days and require frequent recalibration, often using a standard finger prick.^5–7 In addition, implantable CGMS can trigger undesirable immune response, resulting in fibrotic tissue separating the device from blood vessels and surrounding tissue. This separation can lead to inaccuracy, a shorter lifetime, and niches susceptible to infection.^8,9 Hence, new monitoring methods must be investigated.

Erythrocytes functionalized with optical sensors, or erythrosensors, could address the need for a biocompatible, long-term monitoring platform. This approach to blood analyte monitoring relies on isolating erythrocytes, loading them with fluorescent sensors, and injecting the erythrosensors into circulation for analyte monitoring.^10,11 The erythrosensors’ fluorescent signal can be excited and detected noninvasively through the skin. With the goal of developing erythrosensors, erythrocyte functionalization, or loading, must conserve features such as shape, surface morphology, size, and hemoglobin content.

Native erythrocytes (NEs) preserve key properties, such as volume and hemoglobin content as they alter shape to transit the circulatory system. Bovine erythrocytes measure around $5\mu \mathrm{m}$ in diameter and have a life span of 160 days, whereas human erythrocytes are around $8\mu \mathrm{m}$ in diameter and have a life span of 120 days.^12,13 The characteristic biconcave shape maximizes the surface area for oxygen exchange and enables deformation for navigation through the smallest capillaries with deformation depending on the diameter of the capillary and the flow rate. Aging erythrocytes present abnormalities in size and morphology due to changes in the cytoskeleton, membrane, and/or precipitation of hemoglobin. Ultimately, the reticulum endothelial system (RES) takes senescent erythrocytes out of circulation.^14,15 Thus, the “simple” architecture of erythrocytes is, in fact, a complex network of mechanical, chemical, and biological interactions determining cell survival.

Several mechanical, chemical, and electrical techniques have been explored to load carrier erythrocytes for drug delivery and as contrast agents for imaging.¹⁶ Changing erythrocytes’ osmotic conditions or inducing electrical breakdown drives reversible pore formation, but under extreme conditions, it leads to lysis of the membrane.¹⁷ With stable pore formation, diffusion allows entrance and exit through membrane pores as equilibration with the external environment occurs.^18–20

Procedures based on hypotonic conditions have been explored for erythrosensor production.^19,21,22 To date, researchers have studied hypotonic dialysis and hypotonic dilution to produce erythrosensors.^10,11 Variants of the hypotonic dialysis technique, employing a dialysis tubing filled with erythrocytes immersed in a hypotonic buffer, allow cargo diffusion through the dialysis membrane and erythrocyte membrane for entrapment, while the dialysis membrane maintains the erythrocyte and hemoglobin concentrations. Hypotonic dilution consists of diluting erythrocytes in hypotonic solution to create pores and quickly reseal the membrane using a hypertonic buffer at 37°C without the use of dialysis membrane. The change in osmolarity creates pores through the erythrocyte membrane and allows cargo loading as erythrocytes equilibrate with the extracellular environment.²³ These membrane pores allow relatively large molecules to diffuse into the cells and generally lead to $\sim 50\%$ loading efficiency. Previous work has shown that the emission spectrum of erythrosensors loaded with fluorescein isothiocyanate conjugated with glycylglycine (FITC-glygly) was equivalent to the emission by free FITC-glygly and was able to track extracellular pH.¹¹ Hypotonic dilution remains the simplest and fastest loading technique. However, the resulting erythrosensor physical characteristics and cargo location within the erythrocyte remain unknown.^24,25

Electroporation is an alternative loading technique that relies on short, intense electrical charges to create transmembrane pores and allow cargo entrance. Due to the membrane potential difference and reversible electric breakdown, the membrane phospholipid reassembles forming hydrophilic pores.²⁶ This technique is used for drug delivery and transformation/transfection applications because the pore size is tunable through electroporation conditions.^27–29 Zimmermann et al.³⁰ showed data indicating that, although bovine erythrocytes have smaller diameter, they require higher field strength for breakdown than human erythrocytes. At 0°C, the pores are stable while increasing the temperature rapidly reseals the membrane.³¹ Limitations associated with current techniques include low encapsulation efficiency, nonuniform dye loading, and loss of hemoglobin, which affect intrinsic cellular functions.²⁸ Under optimal conditions, erythrocytes loaded using this technique have high cell recovery, maintain their size and shape, and show normal in vivo circulation times.³² These attributes motivated the evaluation of electroporated erythrocytes for use as erythrosensors.

Optimal long-term erythrosensor performance depends on preserving erythrocyte properties while incorporating sensing capabilities. The loss of hemoglobin during the loading process leads to irreversible morphological changes.^32–34 These morphological changes in erythrocytes can subsequently lead to increased stiffness, halting their ability to squeeze through capillaries, and causing clogging as well as cellular removal.¹⁴ Furthermore, reports of carrier erythrocyte cellular modifications, leading to rapid clearance through the RES, a target for drug delivery, and phagocytosis, have limited their implementation in other applications.^14,15,35 Pathological and NEs have been extensively studied using atomic-force microscopy (AFM).^36–38 Despite the advantages of using erythrocytes as carrier systems for optical sensors and drug delivery, little is known about the cellular characteristics of the loaded cells.

Brähler et al.³⁹ found that carrier erythrocytes with magnetite cargo loaded via a hypoosmotic technique had a rugged cross-section profile as evaluated via AFM. This data served as motivation and a guide for continued comparative investigations of the properties of NEs and erythrosensors using AFM. The work described here offers insight into maximizing loading of an optically active cargo, FITC-glygly, into erythrocytes by optimizing and implementing loading protocols. We focus on exploring the loading impact on erythrosensors created via hypotonic dilution. To understand the erythrosensor morphology, loading uniformity, and volume-loading capacity, both confocal microscopy and AFM are used. The cellular properties and the fluorescent signal of two different loading procedures, hypotonic dilution and electroporation, were evaluated (Fig. 1). Work in the drug delivery field using erythrocytes has demonstrated that general loading principles apply across species. For example, studies for the encapsulation of L-asparaginase as a therapeutic molecule in erythrocytes have been carried out in mice, monkeys, and humans.^40–42 Although the specifics of loading change slightly with species, erythrocytes from different species respond to osmolarity changes and electrical charges similarly. This study investigates bovine erythrosensors as a model system for carrying capabilities and identifies detrimental cellular modifications for long-term monitoring.

Fig. 1

Loading of erythrocytes via electroporation and dilution. Isolated erythrocytes were subjected to either electroporation or hypotonic dilution. Electroporation relies on the application of electrical charges to disrupt the membrane potential, allowing cargo entrance and requiring resealing to complete loading. Cells that underwent dilution are lysed and subsequently resealed to encapsulate cargo.

2.

Materials and Methods

3.

Results

Electroporation loading of FITC-glygly into isolated bovine erythrocytes was evaluated under different conditions. Erythrocytes electroporated at 200, 300, and 750 V were found to have average diameters of $4.37\pm 0.54\mu \mathrm{m}$ (average ± stdev), $5.32\pm 0.80\mu \mathrm{m}$, and $5.73\pm 0.88\mu \mathrm{m}$ stdev, respectively (data not shown). Electroporated at 300 V, erythrocytes maintained an average diameter of $4.89\pm 0.49\mu \mathrm{m}$, which lies in the lower margin of the NE cell range. Although erythrocytes loaded using a 750 V amplitude had a larger diameter size, they also demonstrated a variation in size that could account for swollen erythrocytes, lysed erythrocytes, and debris fragments. Increasing the amplitude (200, 300, 400, 750, and 1000 V), and thus the field of strength, decreased the hct of recovered erythrosensors. Erythrosensors loaded via electroporation had the highest fluorescence per cell when four 0.5-ms pulses of 300 V amplitude were applied using a 0.2-cm cuvette, equivalent to an electric field strength of $1.5\mathrm{kV}/\mathrm{cm}$. These conditions were used for the rest of the experiments.

Fluorescence micrographs were used to evaluate the relative cell fluorescence from erythrosensors loaded via hypotonic dilution and electroporation (Fig. 2). Erythrocytes loaded via hypotonic dilution had an average of four times more fluorescence intensity than cells loaded using electroporation ($P<0.05$). Erythrocytes that go through the loading procedures (electroporation or hypotonic dilution) lacking the fluorescent cargo FITC-glygly did not display auto fluorescence. Neither EE nor EH exhibited auto fluorescence associated with the opening of pores via hypotonic dilution or electroporation. A circular outline is seen in the fluorescent and bright-field micrographs for erythrosensors resulting from both techniques, the hypotonic dilution and electroporation. However, the erythrocytes characteristic biconcave shape is not readily evident from the $20\times$ micrograph (Fig. 2). Additionally, fewer erythrocytes were recovered after hypotonic dilution than after electroporation with only 10% to 20% of the initial sample recovered using hypotonic dilution, whereas 40% to 50% of the sample was recovered using electroporation.

Fig. 2

Bovine erythrocytes loaded via either electroporation or dilution and fluorescence per cell revealed as a measure of loading efficiency. Bright-field imaging shown on top and corresponding fluorescence images shown below. Cell’s integrated density, area, and the mean fluorescence of the background were used to evaluate the relative mean fluorescence signal per cell. Bars indicate standard deviation ($*P\le 0.05$).

From the micrographs, the diameter and roundness were evaluated in erythrosensors and compared with the values of NEs (Fig. 3). Additionally, the hemoglobin retained was evaluated in erythrosensors and compared with the values of NEs using spectrophotometry (Fig. 3). Diameters were significantly different among erythrosensors created by electroporation, hypotonic dilution, and NEs as control. The average cell diameter of the NEs measured $6.2\pm 1.13\mu \mathrm{m}$ ($\text{average}\pm \mathrm{stdev}$), significantly greater than the published bovine erythrocytes’ diameter of $5.84\mu \mathrm{m}$.^12,43,44 The erythrocytes, which underwent dilution and electroporation, measured $5.0\pm 0.51\mu \mathrm{m}$ and $5.32\pm 0.80\mu \mathrm{m}$, respectively. In terms of roundness, there was no significant difference between NEs as control and erythrosensors generated using electroporation or hypotonic dilution. The roundness values for electroporated, dilution, and control samples were $0.887\pm 0.108$, $0.908\pm 0.052$, and $0.909\pm 0.099$, respectively. Bovine erythrocytes contain between 8 and $15\mathrm{g}/\mathrm{dL}$ hemoglobin.¹² The hemoglobin in the NEs, as control samples, was estimated between 7 and $9\mathrm{g}/\mathrm{dL}$, which correlates well with the values found in the literature.¹² The average hemoglobin concentration inside erythrosensors loaded via electroporation was $5.63\pm 0.60\mathrm{g}/\mathrm{dL}$. Thus, electroporated erythrocytes lost $\sim 46\%$ of their total hemoglobin on average. The concentration of hemoglobin inside the erythrosensors loaded via hypotonic dilution was $1.00\pm 0.56\mathrm{g}/\mathrm{dL}$. Thus, erythrosensors produced via hypotonic dilution lost $\sim 89\%$ of their total hemoglobin content on average. Although erythrosensors created using electroporation retained higher concentrations of hemoglobin, both hypotonic dilution and electroporation erythrosensors lost significant portions of their hemoglobin. This loss could present a challenge to the in vivo survival of erythrosensors.

Fig. 3

Characterization of erythrosensor size, shape, and hemoglobin retention compared with control NEs. Hemoglobin concentration inside erythrosensors. Absorbance at 540 nm of hemoglobin in sample of lyse control NEs, erythrosensors prepared using the hypotonic dilution and electroporation technique. Bars indicate standard deviation ($*P\le 0.05$, $****P\le 0.0001$).

The low loading efficiency of the electroporation technique would make it difficult to develop an effective in vivo erythrosensor-based measurement system. Thus, confocal microscopy was used to evaluate key population features in the erythrosensors generated using hypotonic dilution. Confocal fluorescent micrographs show nonuniform loading of the sample (Fig. 4). A representative confocal Z-stack created using a step size of $0.39\mu \mathrm{m}$ to evaluate the loading uniformity of the population and of the FITC-glygly entrapment. The sequential images were processed to create the volumetric image and then rotated 90 deg over the $x$-axis to reveal the distribution of FITC-glygly. The 3-D images rendered insight into erythrosensors’ shape and demonstrated volume loading. In addition to the erythrosensors distinctive round shape, the micrographs revealed a population of erythrocytes with $\sim 30\%$ loading efficiency and a nonuniform fluorescence signal.

Fig. 4

Confocal microscopy erythrosensors loaded via hypotonic dilution technique entrapping FITC-glygly. (a) Volume rendering sequence of FITC-glygly-loaded erythrosensors showing their spherical shape. (b) Representative micrographs of erythrosensors exhibiting loading heterogeneity of the resulting population. Bright-field imaging and fluorescence are shown at the top and the bottom, respectively.

To further understand the changes in morphology, AFM was used to study the topography and the cross-section profile of NEs (control) and hypotonic dilution erythrosensors. AFM results show a $10\mu \mathrm{m}\times 10\mu \mathrm{m}$ area and the corresponding topographical image [Fig. 5(a)]. Control erythrocytes exhibit a characteristic shape and dimensions with a toroidal shape with a $5.85\pm 0.18\mu \mathrm{m}$ diameter. The hypotonic dilution erythrosensors showed a corrugated, round, and flat shape with a diameter of $5.30\pm 1.63\mu \mathrm{m}$. Comparing the surface profile of erythrocytes and erythrosensors revealed major morphological changes. The highest peaks detected on the erythrosensors topography average $0.77\pm 0.17\mu \mathrm{m}$ compared with $1.42\pm 0.31\mu \mathrm{m}$ in the control. Erythrosensors, although similar in diameter, had a greater variability in diameter and had a height only half that of normal bovine erythrocytes.

Fig. 5

Erythrosensors’ topography reveals hills and valleys in their surface. (a) The height of the erythrosensors shown in these images lies between 481 and 883 nm. (b) Ra value measuring the mean of the absolute vertical deviation from mean line of the topographical profile. Bars indicate standard deviation ($*P\le 0.05$).

The roughness of the topography of the control erythrocytes and the erythrosensors exhibit differences in the topographical images. The Ra value was found to be greater in erythrosensors [Fig. 5(b)]. The surface roughness of erythrosensors, as measured by the Ra value (0.2 nm), is $\sim 33\%$ rougher than the surface of NEs (0.14 nm). The evidence suggests that erythrosensors loaded via hypotonic dilution exhibit morphological changes that could compromise erythrosensor in vivo life span.

4.

Discussion

In advance of employing erythrocytes as a sensor platform, we explored erythrosensor fluorescent signal, dimensions, morphology, and hemoglobin retention. These are essential features to maintain erythrosensors in circulation for long-term monitoring. Loading procedures were optimized around encapsulation of pH sensitive FITC-glygly in erythrocytes for both hypotonic dilution and electroporation. Comparing these loading techniques, erythrosensors produced via hypotonic dilution resulted in higher loading efficiency. Higher loading of the optical sensor is crucial for providing sufficient optical signal for long-term, noninvasive measurements based on erythrosensors reinfused into circulation. The diameter of electroporated erythrocytes (with and without FITC-glygly) is smaller when compared with NEs likely due to the loss of cell content. This is supported by the fact that erythrocytes electroporated with cargo FITC-glygly (erythrosensors) have a larger diameter than erythrocytes electroporated without FITC-glygly (EE). When loading erythrocytes via electroporation, the cells maintained higher hemoglobin concentration than the dilution method, but still lost approximately half of their total hemoglobin. The loss of hemoglobin in both cases could be an impediment to erythrosensor survival in circulation. This study suggests that hemoglobin levels and cargo loading concentrations are inversely related. These findings are especially useful as a basis to standardize the characterization erythrosensors for use in an erythrosensor-based blood analyte monitoring system.

Erythrocytes loaded via hypotonic dilution and electroporation maintained a rounded shape. Abnormal shapes indicate cytoskeleton or membrane disruption or secondary issues including precipitation of hemoglobin. Thus, the shape of the erythrocyte and erythrosensors can be used as a marker of functionality and integrity of the cell membrane and the cytoskeleton. Our findings (i.e., misshape of erythrosensors and loss of hemoglobin) suggest that an alternative loading to produce erythrosensors that better mimic NEs must be found for long-term monitoring.

One of the attributes of the erythrosensors loaded via hypotonic dilution is the emission of a stronger fluorescent signal when compared with electroporated erythrosensors, which motivated the in-depth study of the hypotonic dilution loading technique. Further characterization of the fluorescence signal emitted by FITC-glygly from erythrosensors resulting from hypotonic dilution revealed a nonuniformly loaded population. The use of an extra centrifugation step to further concentrate loaded erythrosensors was established as a standard for the study of erythrosensors via confocal microscopy and AFM. Isolating erythrosensors with similar fluorescence increases the chances that, for example during AFM topographical images, the erythrosensors studied will be loaded with FITC-glygly. Most importantly, it was demonstrated that the erythrosensors were volume loaded with FITC-glygly. Before this study, the location of the cargo in carrier erythrocytes was not well characterized. Although previous studies suggested the entrapment of cargo, there was no conclusive evidence of volume loading.^10,11,35

Erythrosensors loaded via hypotonic dilution deformed, and their surface topography became rugged. After verifying erythrosensors’ integrity via AFM, it was confirmed that erythrosensors loaded via hypotonic dilution lost the NE architecture consistent with Brähler et al.'s³⁹ findings, in which the cellular changes of carrier erythrocytes were first studied via AFM. The use of AFM to characterize erythrosensors provided a route to explore these changes in conjunction for fluorescence studies.

Here, the characterization of erythrosensors loaded using the hypotonic dilution included the evaluation of size, morphology, hemoglobin retention, and loading uniformity. Decreased size, increased deformations, and rugged topography were some of the erythrosensor attributes found here. The cellular artifacts of carrier erythrocytes and erythrosensors were not well understood and further exploration/optimization of the loading techniques will be required to minimize cellular alterations and potentially increase lifetime circulation as a long-term bloodstream monitoring platform.

5.

Conclusions

Because of their simplicity, biocompatibility, and biodegradability, erythrocytes are an appealing platform for blood-based delivery and monitoring. Erythrocytes depend on a variety of properties including cell size, shape, and hemoglobin content to fulfill their physiological roles. Although sensor functionalized erythrocytes, a.k.a. erythrosensors, offer an intriguing solution for long-term blood analyte monitoring, several factors can influence the yield, integrity, and performance of the erythrosensors. For example, the functionalization process could have deleterious effects on cell structure and/or function. Thus, the cellular properties of erythrosensors will impact their functionality as a long-term monitoring platform.

This study sought deeper insight into cellular changes experienced by erythrosensors loaded via common techniques, hypotonic dilution, and electroporation. Morphological changes in erythrosensors and carrier erythrocytes after hypotonic dilution and electroporation loading were previously not well investigated. This study strove to develop protocols for the characterization of the physical properties of erythrosensors and identify how erythrosensors differ from NEs. Although hypotonic dilution appears to load erythrosensors with sufficient cargo, the identified cellular changes could jeopardize the erythrosensors’ ability to mimic NEs and function as a long-term sensor platform in the bloodstream. From this study, neither hypotonic dilution nor electroporation provide erythrosensors that precisely mimic erythrocytes. Therefore, this study provides a step toward understanding the cellular properties of erythrosensors and standardizing methodologies to assess carrier erythrocytes and erythrosensors.